Treatment of cognitive impairment of hunter syndrome by intrathecal delivery of iduronate-2-sulfatase

ABSTRACT

The present invention, provides a method of treating cognitive impairment of Hunter syndrome. Among other things, the present invention provides a method comprising a step of administering intrathecally to a subject in need of treatment a recombinant iduronate-2-sulfatase (I2S) enzyme at a therapeutically effective dose and an administration interval for a treatment period sufficient to improve, stabilize or reduce declining of one or more cognitive, adaptive, motor, and/or executive functions relative to a control

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/580,027, filed on Dec. 23, 2011; U.S. Provisional Application No.61/590,797, filed on Jan. 25, 2012; U.S. Provisional Application No.61/590,804, filed on Jan. 25, 2012; U.S. Provisional Application No.61/609,173, filed on Mar. 9, 2012; U.S. Provisional Application No.61/734,365, filed on Dec. 6, 2012; the disclosures of each of which areincorporated herein by reference.

BACKGROUND

Hunter syndrome, also known as mucopolysaccharidosis Type II (MPS II),is a lysosomal storage disease caused by deficiency or absence ofenzyme, iduronate-2-sulfatase (I2S). Iduronate-2-sulfatase is involvedin break down and recycle of specific mucopolysaccharides, also known asglycosaminoglycans or GAG. As a result, in Hunter syndrome, GAG buildsup in cells throughout the body, which interferes with the normalfunction of various cells and organs in the body, resulting in a numberof serious symptoms. In many cases of Hunter syndrome, there is often alarge build-up of GAGs in neurons and meninges of affected individuals,leading to various forms of CNS symptoms, impaired cognitive performanceand developmental delays.

Enzyme replacement therapy (ERT) has been used to treat Hunter syndrome.Approved therapy uses intravenous administration of recombinant I2Senzyme. However, intravenously administered enzyme typically does notadequately cross the blood-brain barrier (BBB) into the cells andtissues of the CNS. Therefore, treatment of CNS symptoms of Huntersyndrome has been especially challenging.

SUMMARY

The present invention provides an effective method for treating Huntersyndrome, in particular, Hunter syndrome with cognitive impairment basedon intrathecal administration of recombinant iduronate-2-sulfatase (I2S)enzyme. The present invention is, in part, based on the first-in-humanclinical study demonstrating safety, tolerability and efficacy ofintrathecal delivery of I2S enzyme in Hunter syndrome (MPS II) patients(e.g., from 3-12 years old) with evidence of cognitive impairment. Forexample, as described in the Examples section below, intrathecaladministration of recombinant I2S enzyme was safe, well tolerated andresulted in significant reduction of GAG levels in cerebrospinal fluid(CSF) of the patients in all dose groups including IT dose as low as 1mg per dose. In many cases, the decline of GAG in CSF was evident afterthe first IT dose of I2S enzyme. Since the GAG level in CSF is animportant indicator of pharmacodynamic activity of I2S in theintrathecal compartment, these results demonstrate that intrathecallyadministered I2S has unexpectedly superior pharmacodynamics activity inthe CNS. Consistent with this observation, intrathecally administeredI2S also resulted in stabilization or improvements in cognitiveperformance, including neurocognitive, adaptive and/or executivefunctions, in several patients after receiving only 6 months oftreatment with intrathecal administration of I2S enzyme. Thisfirst-in-human clinical study confirms that intrathecal delivery ofrecombinant I2S enzyme is a safe and effective method for treatingHunter syndrome. In particular, the cognitive data from thisfirst-in-human clinical trial demonstrate that intrathecal delivery ofI2S enzyme may be used to effectively treat CNS symptoms of Huntersyndrome, resulting in stabilization or improvement of cognitiveperformance. It is contemplated that longer duration of treatment inpatients who begin intrathecal therapy early in the trajectory ofneurodevelopmental decline may be particularly effective in treatingcognitive impairment.

Thus, in one aspect, the present invention provides a method of treatingHunters Syndrome comprising a step of administering intrathecally to asubject in need of treatment a recombinant iduronate-2-sulfatase (I2S)enzyme at a therapeutically effective dose and an administrationinterval such that one or more cognitive or developmental abilities areimproved relative to a control. In some embodiments, the presentinvention provides a method of treating Hunter syndrome comprising astep of administering intrathecally to a subject in need of treatment arecombinant iduronate-2-sulfatase (I2S) enzyme at a therapeuticallyeffective dose and an administration interval for a treatment periodsufficient to improve, stabilize or reduce declining of one or morecognitive, adaptive, motor, and/or executive functions relative to acontrol. As used herein, the terms “improve,” “stabilize” or “reduce,”or grammatical equivalents, indicate an assessment or measurement ofcognitive, adaptive, motor, and/or executive functions (e.g., cognitivetest scores) that are relative to a baseline assessment or measurement,such as an assessment or measurement in the same individual prior toinitiation of the treatment, or an assessment or measurement in acontrol individual (or multiple control individuals) in the absence ofthe treatment. A “control individual” is an individual afflicted withHunter syndrome as the individual being treated, who is about the sameage as the individual being treated (to ensure that the stages of thedisease, as well as the stage of childhood development, in the treatedindividual and the control individual(s) are comparable).

In some embodiments, a therapeutically effective dose is or greater thanabout 1 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mgor 100 mg. In particular embodiments, a therapeutically effective doseis or greater than about 10 mg. In particular embodiments, atherapeutically effective dose is or greater than about 30 mg. In someembodiments, a therapeutically effective dose is less than about 50 mg,45 mg, 40 mg, 35 mg, 30 mg, 25 mg, 20 mg, 15 mg, or 10 mg. In particularembodiments, a therapeutically effective dose is less than about 30 mg.In some embodiments, a therapeutically effective dose ranges betweenabout 1-100 mg, about 5-100 mg, about 5-90 mg, about 5-80 mg, about 5-70mg, about 5-60 mg, about 5-60 mg, about 10-100 mg, about 10-90 mg, about10-80 mg, about 10-70 mg, about 10-60 mg, or about 10-50 mg.

In some embodiments, a suitable therapeutically effective dose, onceadministered regularly at the administration interval, results in serumAUC_(ss) of the recombinant I2S enzyme within a range from approximately200,000 min·ng/mL to approximately 1,000,000 min·ng/mL (e.g., fromapproximately 250,000 min·ng/mL to approximately 900,000 min·ng/mL, fromapproximately 300,000 min·ng/mL to approximately 800,000 min·ng/mL, fromapproximately 350,000 min·ng/mL to approximately 700,000 min·ng/mL, fromapproximately 400,000 min·ng/mL to approximately 600,000 min·ng/mL).

In some embodiments, a suitable therapeutically effective dose, onceadministered regularly at the administration interval, results inmaximum serum concentration (C_(max)) of the recombinant I2S enzymewithin a range from approximately 60 to approximately 300 ng/mL (e.g.,from approximately 70 to approximately 250 ng/mL, from approximately 70to approximately 200 ng/mL, from approximately 70 to approximately 150ng/mL, from approximately 80 to approximately 250 ng/mL, fromapproximately 80 to approximately 200 ng/mL, from approximately 80 toapproximately 150 ng/mL, from approximately 90 to approximately 250ng/mL, from approximately 90 to approximately 200 ng/mL, fromapproximately 90 to approximately 150 ng/mL).

In some embodiments, suitable administration interval is weekly, onceevery two weeks, twice a month, once every three weeks, monthly, onceevery two months, once every three months, once every four months, onceevery five months, once every six months, twice a year, once a year, orat a variable interval. As used herein, monthly is equivalent of onceevery four weeks.

In some embodiments, intrathecal administration is through lumbarpuncture. In some embodiments, intrathecal administration is through anOmmaya reservoir. In some embodiments, intrathecal administration isthrough intermittent or continuous access to an implanted intrathecaldrug delivery device (IDDD). In some embodiments, intrathecaladministration is through continuous access to an implanted IDDD for,e.g., greater than 0.1, 0.2, 0.3, 0.4, 0.5, 10, 1.5, 2.0, 2.5, 3.0, 3.5,4.0, 4.5, or 5.0 hours.

In some embodiments, a treatment period is at least 1, 2, 3, 4, 5, 6, 8,10, 12, 18, 24, or more months. In some embodiments, a treatment periodis at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 years or longer. In someembodiments, a treatment period is the life-time of the subject beingtreated.

In some embodiments, the one or more cognitive, adaptive, motor, and/orexecutive functions are assessed by the Differential Ability ScalesSecond Edition (DAS-II). In some embodiments, the DAS-II assessment isby a raw score, cluster score, standardized score, percentile ageequivalent, or developmental quotient. In some embodiments, the DAS-IIassessment is by a general conceptual ability (GCA) score. In someembodiments, the one or more cognitive, adaptive, motor, and/orexecutive functions are assessed by Bayley Scales of Infant DevelopmentVersion III (BSID-III).

In some embodiments, intrathecal administration of the recombinant I2Senzyme results in improved GCA score or BSID-III developmental quotient(DQ) relative to a control (e.g., baseline pre-treatment score). In someembodiments, intrathecal administration of the recombinant I2S enzymeimproves the GCA score or BSID-III developmental quotient by about 5,10, 11, 12, 13, 14, 15, 20, 25, 30 points or more as compared to acontrol (e.g., baseline pre-treatment score). In some embodiments,intrathecal administration of the recombinant I2S enzyme improves theGCA score or BSID-III developmental quotient by about 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50% or more as compared to a control (e.g.,baseline pre-treatment score). In some embodiments, the improved GCAscore or BSID-1l developmental quotient is within the range of 85-105within the range of approximately 70-105 (e.g., approximately 75-105,75-100, 70-100, 70-95, 70-90, 75-105, 75-100, 75-95, 75-90, 80-105,80-100, 80-95, 80-90, 85-105, 85-100, or 85-95). In some embodiments,the improved GCA score or BSID-III developmental quotient is or greaterthan about 70, 75, 80, 85, 86, 87, 88, 89, 90, 95, 96, 97, 98, 99, 100,101, 102, 103, 104, or 105 points. In some embodiments, the GCA score orBSID-II developmental quotient is measured after a treatment period ofor longer than 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 18 months. In someembodiments, intrathecal administration of the recombinant I2S enzymemaintains the improved GCA score or BSID-III developmental quotient fora period of or longer than 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, or36 months. As used herein, maintaining the GCA score or BSID-IIIdevelopmental quotient means the change of GCA score or BSID-IIIdevelopmental quotient is less than 10, 9, 8, 7, 6, or 5 points within aperiod of 3, 6, 8, 10, 12 months or the change of the GCA score orBSID-III developmental quotient over a period of 3, 6, 8, 10, 12 monthsis within 20%, 15%, 10%, 5% of the mean over such period.

In some embodiments, intrathecal administration of the recombinant I2Senzyme results in stabilization of the GCA score or BSID-IIIdevelopmental quotient relative to a control (e.g., baselinepre-treatment score). In some embodiments, intrathecal administration ofthe recombinant I2S enzyme results in stabilization of the GCA score orBSID-III developmental quotient relative to the baseline pre-treatmentscore after a treatment period of or longer than 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 18 months, or 1, 2, 3, 4, 5, 10 years. As used herein,stabilization of the GCA score or BSID-III developmental quotient meansthe change of GCA score or BSID-III developmental quotient from thebaseline is less than 10, 9, 8, 7, 6, or 5 points within 3, 6, 8, 10, 12months or the change of the GCA score or BSID-III developmental quotientover a period of 3, 6, 8, 10, 12 months within 20%, 15%, 10%, 5% of themean over such period. In some cases, stabilization of the GCA score orBSID-III developmental quotient means the change of GCA score orBSID-III developmental quotient from the baseline is less than 20%, 15%,10%, 5% within 3, 6, 8, 10, 12 months. In some embodiments, intrathecaladministration of the recombinant I2S enzyme results in stabilization ofthe GCA score or BSID-III developmental quotient following the initialdeclining of the GCA score or BSID-III developmental quotient. Forexample, stabilization may follow after no less than 40%, 35%, 30%, 25%,20%, 15%, or 10% declining of the GCA score or BSID-III developmentalquotient from the baseline. In some embodiments, intrathecaladministration of the recombinant I2S enzyme stabilizes the GCA score orBSID-III developmental quotient for a period of or longer than 3, 6, 9,12, 15, 18, 21, 24, 27, 30, 33, or 36 months. In some embodiments,intrathecal administration of the recombinant I2S enzyme stabilizes theGCA score or BSID-III developmental quotient for a period of 3-36 months(e.g., 3-33, 3-30, 3-27, 3-24, 3-21, 3-18, 3-15, 3-12, 3-9, 3-6, 6-36,6-33, 6-30, 6-27, 6-24, 6-21, 6-18, 6-15, 6-12, 6-9 months).

In some embodiments, intrathecal administration of the recombinant I2Senzyme results in reduced declining of the GCA score or BSID-IIIdevelopmental quotient. In some embodiments, intrathecal administrationof the recombinant I2S enzyme results in the annual decline of the GCAscore or BSID-III developmental quotient less than about 20, 19, 18, 17,16, 15, 14, 13, 12, 11, or 10 points. In some embodiments, intrathecaladministration of the recombinant I2S enzyme results in the annualdecline of the GCA score or BSID-III developmental quotient less thanabout 40%, 35%, 30%, 25%, 20%, 15%, or 10%. In some embodiments, reduceddeclining of the GCA score or BSID-III developmental quotient isachieved after a treatment period of or longer than 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 18 months, or 1, 2, 3, 4, 5, 10 years.

In some embodiments, intrathecal administration of the recombinant I2Senzyme further results in improvement or stabilization of one or moreadaptive functions assessed by the Scales of IndependentBehavior-Revised (SIB-R). In some embodiments, intrathecaladministration of the recombinant I2S enzyme further results inimprovement or stabilization of one or more executive functions assessedby the Behavior Rating Inventory of Executive Function® (BRIEF®).

The present invention may be used to treat a subject of various ages. Insome embodiments, a subject being treated is at least 6 moths old, 12months old, at least 18 months old, 2 years old, 2.5 years old, 3 yearsold, 3.5 years old, 4 years old, 4.5 years old, or 5 years old. In someembodiments, a subject being treated is younger than 5, 4.5, 4, 3.5, 3,2.5, 2, or 1.5 years old. In some embodiments, a subject being treatedis younger than 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 month old. Insome embodiments, a subject being treated is younger than 29, 28, 27,26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9,8, 7, 6, 5, 4, 3, 2, 1 day(s) old. In some embodiments, a subject beingtreated is within the age range of 0 months-8 years, 3 months-8 years, 6months-8 years, 8 months-8 years, 1 year-8 years, 3 months-7 years, 6months-7 years, 8 months-7 years, 1 year-7 years, 3 months-6 years, 6months-6 years, 8 months-6 years, 1 year-6 years, 3 months-5 years, 6months-5 years, 8 months-5 years, 1 year-5 years, 3 months-4 years, 6months-4 years, 8 months-4 years, 1 year-4 years, 3 months-3 years, 6months-3 years, 8 months-3 years, 1 year-3 years, 3 months-2 years, 6months-2 years, 8 months-2 years, or 1 year-2 years, 3 months-1 year, 6months-1 year, or 8 months-1 year old.

In some embodiments, a subject being treated has cognitive impairment.In some embodiments, a subject being treated has a GCA score or BSID-IIIdevelopmental quotient less than 100, 90, 80, 70, 60, 50, 40, 30, 20,15, 10 or not testable before the treatment. In some embodiments, asubject being treated has a GCA score or BSID-III developmental quotientdeclined from normal baseline less than about 40%, 35%, 30%, 25%, 20%,15%, or 10% before the treatment. In some embodiments, a subject beingtreated has a GCA score or BSID-III developmental quotient rangingbetween about 60-100 (e.g., about 60-95, 60-90, 60-85, 60-80, 60-75,60-70, 70-100, 70-95, 70-90, 70-85, 70-80, 80-100, 80-95, 80-90) beforethe treatment.

In various embodiments, intrathecal administration is performed inconjunction with intravenous administration of the recombinant I2Senzyme. In some embodiments, intravenous administration of therecombinant I2S enzyme is weekly. In some embodiments, the intravenousadministration of the recombinant I2S enzyme is weekly except the weekwhen the intrathecal administration is performed. In some embodiments,the intravenous administration of the recombinant I2S enzyme isbiweekly, once every three weeks, monthly, twice a month, once everytwo, three, four, five, or six months. In some embodiments, theintravenous administration of the recombinant I2S enzyme is at a dose ofabout 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 mg/kg bodyweight. In some embodiments, the intravenous administration of therecombinant I2S enzyme is at a dose of about 0.5 mg/kg body weight.

In some embodiments, the dose and/or administration interval forintrathecal and/or intravenous administration may be adjusted (e.g.,increasing or decreasing) based on the GCA, BSID-II, SIB-R, and/or BRIEFscore

In various embodiments, intrathecal administration according to theinvention results in no serious adverse effects in the subject. Invarious embodiments, intrathecal administration according to theinvention does not require an immunosuppressant.

In particular embodiments, the present invention provides a method oftreating Hunter syndrome comprising administering intrathecally to asubject in need of treatment a recombinant iduronate-2-sulfatase (I2S)enzyme at a first therapeutically effective dose and administeringintravenously to the subject the recombinant I2S enzyme at a secondtherapeutically effective dose for a treatment period sufficient toimprove, stabilize or reduce declining of one or more cognitive,adaptive, motor, and/or executive functions relative to a control. Insome embodiments, the intrathecal administration is monthly. In someembodiments, the intravenous administration is weekly.

In another aspect, the present invention provides a method of treatingHunter syndrome comprising a step of administering intrathecally to asubject in need of treatment a recombinant iduronate-2-sulfatase (I2S)enzyme at a therapeutically effective dose and an administrationinterval for a treatment period sufficient to decrease glycosaminoglycan(GAG) level in the cerebrospinal fluid (CSF) relative to a control. Asused herein, the term “decrease,” or equivalent such as “reduce,” orgrammatical equivalents, indicate a measurement of GAG level that isrelative to a baseline measurement, such as a measurement in the sameindividual prior to initiation of the treatment, or a measurement in acontrol individual (or multiple control individuals) in the absence ofthe treatment. A “control individual” is an individual afflicted withHunter syndrome as the individual being treated, who is about the sameage as the individual being treated (to ensure that the stages of thedisease in the treated individual and the control individual(s) arecomparable).

In some embodiments, a therapeutically effective dose is or greater thanabout 1 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mgor 100 mg. In particular embodiments, a therapeutically effective doseis or greater than about 10 mg. In particular embodiments, atherapeutically effective dose is or greater than about 30 mg. In someembodiments, a therapeutically effective dose is less than about 50 mg,45 mg, 40 mg, 35 mg, 30 mg, 25 mg, 20 mg, 15 mg, or 10 mg. In particularembodiments, a therapeutically effective dose is less than about 30 mg.In some embodiments, a therapeutically effective dose ranges betweenabout 1-100 mg, about 5-100 mg, about 5-90 mg, about 5-80 mg, about 5-70mg, about 5-60 mg, about 5-60 mg, about 10-100 mg, about 10-90 mg, about10-80 mg, about 10-70 mg, about 10-60 mg, or about 10-50 mg.

In some embodiments, a suitable therapeutically effective dose, onceadministered regularly at the administration interval, results in serumAUC_(ss) of the recombinant I2S enzyme within a range from approximately200,000 min·ng/mL to approximately 1,000,000 min·ng/mL (e.g., fromapproximately 250,000 min·ng/mL to approximately 900,000 min·ng/mL, fromapproximately 300,000 min·ng/mL to approximately 800,000 min·ng/mL, fromapproximately 350,000 min·ng/mL to approximately 700,000 min·ng/mL, fromapproximately 400,000 min·ng/mL to approximately 600,000 min·ng/mL).

In some embodiments, a suitable therapeutically effective dose, onceadministered regularly at the administration interval, results inmaximum serum concentration (C_(max)) of the recombinant I2S enzymewithin a range from approximately 60 to approximately 300 ng/mL (e.g.,from approximately 70 to approximately 250 ng/mL, from approximately 70to approximately 200 ng/mL, from approximately 70 to approximately 150ng/mL, from approximately 80 to approximately 250 ng/mL, fromapproximately 80 to approximately 200 ng/mL, from approximately 80 toapproximately 150 ng/mL, from approximately 90 to approximately 250ng/mL, from approximately 90 to approximately 200 ng/mL, fromapproximately 90 to approximately 150 ng/mL).

In some embodiments, suitable administration interval is weekly, onceevery two weeks, twice a month, once every three weeks, monthly, onceevery two months, once every three months, once every four months, onceevery five months, once every six months, twice a year, once a year, orat a variable interval. As used herein, monthly is equivalent of onceevery four weeks.

In some embodiments, intrathecal administration is through lumbarpuncture. In some embodiments, intrathecal administration is through anOmmaya reservoir. In some embodiments, intrathecal administration isthrough intermittent or continuous access to an implanted intrathecaldrug delivery device (IDDD). In some embodiments, intrathecaladministration is through continuous access to an implanted IDDD for,e.g., greater than 0.1, 0.2, 0.3, 0.4, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0,3.5, 4.0, 4.5, or 5.0 hours

In some embodiments, a treatment period is at least 1, 2, 3, 4, 5, 6, 8,10, 12, 18, 24, or more months. In some embodiments, a treatment periodis at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 years or longer. In someembodiments, a treatment period is the life-time of the subject beingtreated.

In some embodiments, intrathecal administration of the recombinant I2Senzyme results in the GAG level in the CSF lower than about 1000 ng/ml(e.g., lower than about 900 ng/ml, 800 ng/ml, 700 ng/ml, 600 ng/ml, 500ng/ml, 400 ng/ml, 300 ng/ml, 200 ng/ml, 100 ng/ml, 50 ng/ml, 10 ng/ml,or 1 ng/ml).

In some embodiments, a subject being treated is at least 6 months old,12 months old, 18 months old, 2 years old, 2.5 years old, 3 years old,3.5 years old, 4 years old, 4.5 years old, or 5 years old. In someembodiments, a subject being treated is younger than 5, 4.5, 4, 3.5, 3,2.5, 2, or 1.5 years old. In some embodiments, a subject being treatedis younger than 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 month old. Insome embodiments, a subject being treated is younger than 29, 28, 27,26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9,8, 7, 6, 5, 4, 3, 2, 1 day(s) old. In some embodiments, the subject inneed of treatment has a GAG level in the CSF greater than about 300,400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 ng/ml before thetreatment.

In various embodiments, intrathecal administration is performed inconjunction with intravenous administration of the recombinant I2Senzyme. In some embodiments, intravenous administration of therecombinant I2S enzyme is weekly. In some embodiments, the intravenousadministration of the recombinant I2S enzyme is weekly except the weekwhen the intrathecal administration is performed. In some embodiments,the intravenous administration of the recombinant I2S enzyme isbiweekly, once every three weeks, monthly, twice a month, once everytwo, three, four, five, or six months. In some embodiments, theintravenous administration of the recombinant I2S enzyme is at a dose ofabout 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 mg/kg bodyweight. In some embodiments, the intravenous administration of therecombinant I2S enzyme is at a dose of about 0.5 mg/kg body weight

In various embodiments, a method according to the present inventionfurther comprises a step of adjusting the dose and/or administrationinterval for intrathecal and/or intravenous administration based on theGAG level in the CSF. In some embodiments, the step of adjustingcomprises increasing the therapeutic effective dose for intrathecaladministration if the GAG level in the CSF fails to decrease relative tothe control after 6, 5, 4, or 3 doses. In some embodiments, the step ofadjusting comprises increasing the therapeutic effective dose forintrathecal administration if the GAG level in the CSF fails to decreaserelative to the control after 4 doses.

In various embodiments, intrathecal administration according to theinvention results in no serious adverse effects in the subject. Invarious embodiments, intrathecal administration according to theinvention does not require an immunosuppressant.

In particular embodiments, the present invention provides a method oftreating Hunter syndrome comprising administering intrathecally to asubject in need of treatment a recombinant iduronate-2-sulfatase (I2S)enzyme at a first therapeutically effective dose and administeringintravenously to the subject the recombinant I2S enzyme at a secondtherapeutically effective dose for a treatment period sufficient todecrease glycosaminoglycan (GAG) level in the cerebrospinal fluid (CSF)relative to a control. In some embodiments, the intrathecaladministration is monthly. In some embodiments, the intravenousadministration is weekly.

In some embodiments, the present invention provides a recombinantiduronate-2-sulfatase (I2S) enzyme for use in a method of treatingHunter Syndrome wherein the method comprises a step of administeringintrathecally to a subject in need of treatment the recombinant I2Senzyme at a therapeutically effective dose and an administrationinterval for a treatment period sufficient to improve, stabilize orreduce declining of one or more cognitive, adaptive, motor, and/orexecutive functions relative to a control.

In some embodiments, the present invention provides use of a recombinantiduronate-2-sulfatase (I2S) enzyme in the manufacture of a medicamentfor treating Hunter Syndrome wherein the treatment comprises a step ofadministering intrathecally to a subject in need of treatment therecombinant I2S enzyme at a therapeutically effective dose and anadministration interval for a treatment period sufficient to improve,stabilize or reduce declining of one or more cognitive, adaptive, motor,and/or executive functions relative to a control.

Various treatment embodiments described herein are suitable for the useof a recombinant I2S enzyme

In some embodiments, the treatment suitable for the use of a recombinantI2S enzyme comprises administering intrathecally to a subject in need oftreatment the recombinant I2S enzyme at a first therapeuticallyeffective dose; and administering intravenously to the subject therecombinant I2S enzyme at a second therapeutically effective dose for atreatment period sufficient to improve, stabilize or reduce declining ofone or more cognitive, adaptive, motor, and/or executive functionsrelative to a control.

In some embodiments, the present invention provides a recombinantiduronate-2-sulfatase (I2S) enzyme for use in a method of treatingHunter syndrome comprising administering intrathecally to a subject inneed of treatment the recombinant I2S enzyme at a first therapeuticallyeffective dose; and administering intravenously to the subject therecombinant I2S enzyme at a second therapeutically effective dose for atreatment period sufficient to improve, stabilize or reduce declining ofone or more cognitive, adaptive, motor, and/or executive functionsrelative to a control.

In some embodiments, the present invention relates to use of arecombinant iduronate-2-sulfatase (I2S) enzyme in the manufacture of amedicament for treating Hunter syndrome wherein the treatment comprisesadministering intrathecally to a subject in need of treatment therecombinant I2S enzyme at a first therapeutically effective dose; andadministering intravenously to the subject the recombinant I2S enzyme ata second therapeutically effective dose for a treatment periodsufficient to improve, stabilize or reduce declining of one or morecognitive, adaptive, motor, and/or executive functions relative to acontrol.

In some embodiments, the present invention provides a recombinantiduronate-2-sulfatase (I2S) enzyme for use in a method of treatingHunter Syndrome wherein the method comprises a step of administeringintrathecally to a subject in need of treatment the recombinant I2Senzyme at a therapeutically effective dose and an administrationinterval for a treatment period sufficient to decrease glycosaminoglycan(GAG) level in the cerebrospinal fluid (CSF) relative to a control.

In some embodiments, the present invention relates to use of arecombinant iduronate-2-sulfatase (I2S) enzyme in the manufacture of amedicament for treating Hunter Syndrome wherein the treatment comprisesa step of administering intrathecally to a subject in need of treatmentthe recombinant 1S enzyme at a therapeutically effective dose and anadministration interval for a treatment period sufficient to decreaseglycosaminoglycan (GAG) level in the cerebrospinal fluid (CSF) relativeto a control. Various treatment embodiments described herein aresuitable for the use of a recombinant I2S enzyme.

In some embodiments, the treatment suitable for the use of a recombinantI2S enzyme comprises administering intrathecally to a subject in need oftreatment the recombinant I2S enzyme at a first therapeuticallyeffective dose; and administering intravenously to the subject therecombinant I2S enzyme at a second therapeutically effective dose for atreatment period sufficient to decrease glycosaminoglycan (GAG) level inthe cerebrospinal fluid (CSF) relative to a control

In some embodiments, the present invention provides a recombinantiduronate-2-sulfatase (I2S) enzyme for use in a method of treatingHunter syndrome comprising administering intrathecally to a subject inneed of treatment the recombinant I2S enzyme at a first therapeuticallyeffective dose; and administering intravenously to the subject therecombinant I2S enzyme at a second therapeutically effective dose, for atreatment period sufficient to decrease glycosaminoglycan (GAG) level inthe cerebrospinal fluid (CSF) relative to a control.

In some embodiments, the present invention relates to use of arecombinant iduronate-2-sulfatase (I2S) enzyme in the manufacture of amedicament for treating Hunter syndrome, the treatment comprisingadministering intrathecally to a subject in need of treatment therecombinant I2S enzyme at a first therapeutically effective dose; andadministering intravenously to the subject the recombinant I2S enzyme ata second therapeutically effective dose, for a treatment periodsufficient to decrease glycosaminoglycan (GAG) level in thecerebrospinal fluid (CSF) relative to a control.

In some embodiments, the method, enzyme for use, or use described hereinis for treating a subject has cognitive impairment. In some embodiments,a subject being treated has a GCA score or BSID-III developmentalquotient less than 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, 10 or nottestable before the treatment. In some embodiments, a subject beingtreated has a GCA score or BSID-III developmental quotient declined fromnormal baseline less than about 40%, 35%, 30%, 25%, 20%, 15%, or 10%before the treatment. In some embodiments, a subject being treated has aGCA score or BSID-III developmental quotient ranging between about60-100 (e.g., about 60-95, 60-90, 60-85, 60-80, 60-75, 60-70, 70-100,70-95, 70-90, 70-85, 70-80, 80-100, 80-95, 80-90) before the treatment.

In some embodiments, the method, enzyme for use, or use described hereinis for stabilizing or improving cognitive performance in a subjecthaving Hunter Syndrome.

In some embodiments, the present invention provides a recombinantiduronate-2-sulfatase (I2S) enzyme for use in a method of stabilizing orimproving cognitive performance in a subject having Hunter Syndromewherein the method comprises a step of administering intrathecally to asubject in need of treatment the recombinant I2S enzyme at atherapeutically effective dose and an administration interval for atreatment period sufficient to improve, stabilize or reduce declining ofone or more cognitive, adaptive, motor, and/or executive functionsrelative to a control.

In some embodiments, the present invention provides a recombinantiduronate-2-sulfatase (I2S) enzyme for use in a method of stabilizing orimproving cognitive performance in a subject having Hunter Syndromewherein the method comprises a step of administering intrathecally to asubject in need of treatment the recombinant I2S enzyme at atherapeutically effective dose and an administration interval for atreatment period sufficient to decrease glycosaminoglycan (GAG) level inthe cerebrospinal fluid (CSF) relative to a control.

As used in this application, the terms “about” and “approximately” areused as equivalents. Any numerals used in this application with orwithout about/approximately are meant to cover any normal fluctuationsappreciated by one of ordinary skill in the relevant art.

Other features, objects, and advantages of the present invention areapparent in the detailed description that follows. It should beunderstood, however, that the detailed description, while indicatingembodiments of the present invention, is given by way of illustrationonly, not limitation. Various changes and modifications within the scopeof the invention will become apparent to those skilled in the art fromthe detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings are for illustration purposes only, not for limitation.

FIGS. 1-4 illustrates exemplary modeling of I2S in serum and CSF.

FIG. 5 illustrates exemplary allometrically scaled populationpharmacokinetic parameters for IT-I2S delivery in pediatric subjects,after correcting for differences between non-human primates and childrenusing a brain and body weight.

FIG. 6 illustrates exemplary body weight scaled I2S serum concentrationsvs. scaled time, in both pediatric subjects and monkeys following IT-Ldosing.

FIG. 7 illustrates the observed vs predicted serum concentration profileof I2S in pediatric subjects, following a single IV infusion.

FIG. 8 illustrates the observed vs predicted serum concentration profileof I2S in pediatric subjects, following a single 10 mg IT-Ladministration.

FIG. 9 illustrates the observed vs predicted serum concentration profileof I2S in pediatric subjects following, a single 10 mg IT-Ladministration. The exemplary concentration profile for both theobserved and predicted are shown, with or without correction for brainand body weight.

FIG. 10 illustrates I2S sampling in serum and CSF of pediatric patientsover various time-points and parameters.

FIG. 11 illustrates the projected I2S serum concentration level in ahuman subject following IT administration at 1, 10, 30 and 100 mg usinga Human model.

FIG. 12 illustrates the projected I2S serum concentration level in ahuman subject following IT administration at 1, 10, 30 and 100 mg usingan Allometric model.

FIG. 13 demonstrates exemplary mean serum I2S concentration-timeprofiles of patients in the first arm at week 3.

FIG. 14 demonstrates exemplary mean serum I2S concentration-timeprofiles of patients in the first arm at week 23.

FIG. 15 demonstrates exemplary mean serum I2S concentration-timeprofiles of patients at week 3, week 23 and month 19

FIG. 16 describes exemplary cerebrospinal fluid levels of GAG inpatients treated with 10 mg of IT recombinant I2S over a 36 monthperiod.

FIG. 17 describes exemplary cerebrospinal fluid levels of GAG inpatients treated with 30 mg of IT recombinant I2S over a 15 monthperiod.

FIG. 18 demonstrates exemplary cerebrospinal fluid levels of GAG inclinical trial patients treated with 1, 10 or 30 mg of I2S over 27weeks, as compared to control.

FIG. 19 illustrates exemplary individual patient SIB-R Broad standardIndependence standard scoreby chronological age.

FIG. 20 illustrates additional exemplary instruments for assessingcognitive performance.

FIG. 21 illustrates the subtests of the DAS-II.

DEFINITIONS

In order for the present invention to be more readily understood,certain terms are first defined below. Additional definitions for thefollowing terms and other terms are set forth throughout thespecification.

Approximately or about: As used herein, the term “approximately” or“about,” as applied to one or more values of interest, refers to a valuethat is similar to a stated reference value. In certain embodiments, theterm “approximately” or “about” refers to a range of values that fallwithin 25%, 20%, 19%, 18%, 17% 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%,8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greaterthan or less than) of the stated reference value unless otherwise statedor otherwise evident from the context (except where such number wouldexceed 100% of a possible value).

Amelioration: As used herein, the term “amelioration” is meant theprevention, reduction or palliation of a state, or improvement of thestate of a subject. Amelioration includes, hut does not require completerecovery or complete prevention of a disease condition. In someembodiments, amelioration includes increasing levels of relevant proteinor its activity that is deficient in relevant disease tissues.

Biologically active: As used herein, the phrase “biologically active”refers to a characteristic of any agent that has activity in abiological system, and particularly in an organism. For instance, anagent that, when administered to an organism, has a biological effect onthat organism, is considered to be biologically active. In particularembodiments, where a protein or polypeptide is biologically active, aportion of that protein or polypeptide that shares at least onebiological activity of the protein or polypeptide is typically referredto as a “biologically active” portion.

Bulking agent: As used herein, the term “bulking agent” refers to acompound which adds mass to the lyophilized mixture and contributes tothe physical structure of the lyophilized cake (e.g., facilitates theproduction of an essentially uniform lyophilized cake which maintains anopen pore structure). Exemplary bulking agents include mannitol,glycine, sodium chloride, hydroxyethyl starch, lactose, sucrose,trehalose, polyethylene glycol and dextran.

Cation-independent mannose-6-phosphate receptor (CI-MPR): As usedherein, the term “cation-independent mannose-6-phosphate receptor(CI-MPR)” refers to a cellular receptor that binds mannose-6-phosphate(M6P) tags on acid hydrolase precursors in the Golgi apparatus that aredestined for transport to the lysosome. In addition tomannose-6-phosphates, the CI-MPR also binds other proteins includingIGF-II. The CI-MPR is also known as “M6P/IGF-II receptor,”“CI-MPR/IGF-II receptor,” “IGF-II receptor” or “IGF2 Receptor.” Theseterms and abbreviations thereof are used interchangeably herein.

Concurrent immunosuppressant therapy: As used herein, the term“concurrent immunosuppressant therapy” includes any immunosuppressanttherapy used as pre-treatment, preconditioning or in parallel to atreatment method,

Diluent: As used herein, the term “diluent” refers to a pharmaceuticallyacceptable (e.g., safe and non-toxic for administration to a human)diluting substance useful for the preparation of a reconstitutedformulation. Exemplary diluents include sterile water, bacteriostaticwater for injection (BWFI), a pH buffered solution (e.g.phosphate-buffered saline), sterile saline solution, Ringer's solutionor dextrose solution.

Dosage form: As used herein, the terms “dosage form” and “unit dosageform” refer to a physically discrete unit of a therapeutic protein forthe patient to be treated. Each unit contains a predetermined quantityof active material calculated to produce the desired therapeutic effect.It will be understood, however, that the total dosage of the compositionwill be decided by the attending physician within the scope of soundmedical judgment.

Enzyme replacement therapy (ERT): As used herein, the term “enzymereplacement therapy (ERT)” refers to any therapeutic strategy thatcorrects an enzyme deficiency by providing the missing enzyme. In someembodiments, the missing enzyme is provided by intrathecaladministration. In some embodiments, the missing enzyme is provided byinfusing into the bloodstream. Once administered, enzyme is taken up bycells and transported to the lysosome, where the enzyme acts toeliminate material that has accumulated in the lysosomes due to theenzyme deficiency. Typically, for lysosomal enzyme replacement therapyto be effective, the therapeutic enzyme is delivered to lysosomes in theappropriate cells in target tissues where the storage defect ismanifest.

Improve, increase, or reduce: As used herein, the terms “improve,”“increase” or “reduce,” or grammatical equivalents, indicate values thatare relative to a baseline measurement, such as a measurement in thesame individual prior to initiation of the treatment described herein,or a measurement in a control individual (or multiple controlindividuals) in the absence of the treatment described herein. A“control individual” is an individual afflicted with the same form oflysosomal storage disease as the individual being treated, who is aboutthe same age as the individual being treated (to ensure that the stagesof the disease in the treated individual and the control individual(s)are comparable).

Individual, subject, patient: As used herein, the terms “subject,”“individual” or “patient” refer to a human or a non-human mammaliansubject. The individual (also referred to as “patient” or “subject”)being treated is an individual (fetus, infant, child, adolescent, oradult human) suffering from a disease.

Intrathecal administration: As used herein, the term “intrathecaladministration” or “intrathecal injection” refers to an injection intothe spinal canal (intrathecal space surrounding the spinal cord).Various techniques may be used including, without limitation, lateralcerebroventricular injection through a burrhole or cisternal or lumbarpuncture or the like. In some embodiments, “intrathecal administration”or “intrathecal delivery” according to the present invention refers toIT administration or delivery via the lumbar area or region, i.e.,lumbar IT administration or delivery. As used herein, the term “lumbarregion” or “lumbar area” refers to the area between L4-L5, L3-L4, L2-L3,and/or L2-S1 regions of the spine.

Polypeptide: As used herein, a “polypeptide”, generally speaking, is astring of at least two amino acids attached to one another by a peptidebond. In some embodiments, a polypeptide may include at least 3-5 aminoacids, each of which is attached to others by way of at least onepeptide bond. Those of ordinary skill in the art will appreciate thatpolypeptides sometimes include “non-natural” amino acids or otherentities that nonetheless are capable of integrating into a polypeptidechain, optionally.

Replacement enzyme: As used herein, the term “replacement enzyme” refersto any enzyme that can act to replace at least in part the deficient ormissing enzyme in a disease to be treated. In some embodiments, the term“replacement enzyme” refers to any enzyme that can act to replace atleast in part the deficient or missing lysosomal enzyme in a lysosomalstorage disease to be treated. In some embodiments, a replacement enzymeis capable of reducing accumulated materials in mammalian lysosomes orthat can rescue or ameliorate one or more lysosomal storage diseasesymptoms. Replacement enzymes suitable for the invention include bothwild-type or modified lysosomal enzymes and can be produced usingrecombinant and synthetic methods or purified from nature sources. Areplacement enzyme can be a recombinant, synthetic, gene-activated ornatural enzyme.

Soluble: As used herein, the term “soluble” refers to the ability of atherapeutic agent to form a homogenous solution. In some embodiments,the solubility of the therapeutic agent in the solution into which it isadministered and by which it is transported to the target site of action(e.g., the cells and tissues of the brain) is sufficient to permit thedelivery of a therapeutically effective amount of the therapeutic agentto the targeted site of action. Several factors can impact thesolubility of the therapeutic agents. For example, relevant factorswhich may impact protein solubility include ionic strength, amino acidsequence and the presence of other co-solubilizing agents or salts(e.g., calcium salts). In some embodiments, the pharmaceuticalcompositions are formulated such that calcium salts are excluded fromsuch compositions. In some embodiments, therapeutic agents in accordancewith the present invention are soluble in its correspondingpharmaceutical composition. It will be appreciated that, while isotonicsolutions are generally preferred for parenterally administered drugs,the use of isotonic solutions may limit adequate solubility for sometherapeutic agents and, in particular some proteins and/or enzymes.Slightly hypertonic solutions (e.g., up to 175 mM sodium chloride in 5mM sodium phosphate at pH 7.0) and sugar-containing solutions (e.g., upto 2% sucrose in 5 mM sodium phosphate at pH 7.0) have been demonstratedto be well tolerated in monkeys. For example, the most common approvedCNS bolus formulation composition is saline (150 mM NaCl in water).

Stability: As used herein, the term “stable” refers to the ability ofthe therapeutic agent (e.g., a recombinant enzyme) to maintain itstherapeutic efficacy (e.g., all or the majority of its intendedbiological activity and/or physiochemical integrity) over extendedperiods of time. The stability of a therapeutic agent, and thecapability of the pharmaceutical composition to maintain stability ofsuch therapeutic agent, may be assessed over extended periods of time(e.g., for at least 1, 3, 6, 12, 18, 24, 30, 36 months or more). Ingeneral, pharmaceutical compositions described herein have beenformulated such that they are capable of stabilizing, or alternativelyslowing or preventing the degradation, of one or more therapeutic agentsformulated therewith (e.g., recombinant proteins). In the context of aformulation a stable formulation is one in which the therapeutic agenttherein essentially retains its physical and/or chemical integrity andbiological activity upon storage and during processes (such asfreeze/thaw, mechanical mixing and lyophilization). For proteinstability, it can be measure by formation of high molecular weight (HMW)aggregates, loss of enzyme activity, generation of peptide fragments andshift of charge profiles.

Subject: As used herein, the term “subject” means any mammal, includinghumans. In certain embodiments of the present invention the subject isan adult, an adolescent or an infant. Also contemplated by the presentinvention are the administration of the pharmaceutical compositionsand/or performance of the methods of treatment in-utero.

Substantial homology: The phrase “substantial homology” is used hereinto refer to a comparison between amino acid or nucleic acid sequences.As will be appreciated by those of ordinary skill in the art, twosequences are generally considered to be “substantially homologous” ifthey contain homologous residues in corresponding positions. Homologousresidues may be identical residues. Alternatively, homologous residuesmay be non-identical residues will appropriately similar structuraland/or functional characteristics. For example, as is well known bythose of ordinary skill in the art, certain amino acids are typicallyclassified as “hydrophobic” or “hydrophilic” amino acids, and/or ashaving “polar” or “non-polar” side chains Substitution of one amino acidfor another of the same type may often be considered a “homologous”substitution.

As is well known in this art, amino acid or nucleic acid sequences maybe compared using any of a variety of algorithms, including thoseavailable in commercial computer programs such as BLASTN for nucleotidesequences and BLASTP, gapped BLAST, and PSI-BLAST for amino acidsequences. Exemplary such programs are described in Altschul, et al.,Basic local alignment search tool, J. Mol. Biol., 215(3): 403-410, 1990;Altschul, et al., Methods in Enzymology; Altschul, et al., “Gapped BLASTand PSI-BLAST: a new generation of protein database search programs”,Nucleic Acids Res. 25:3389-3402, 1997; Baxevanis, et al.,Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins,Wiley, 1998; and Misener, et al., (eds.), Bioinformatics Methods andProtocols (Methods in Molecular Biology, Vol. 132), Humana Press, 1999.In addition to identifying homologous sequences, the programs mentionedabove typically provide an indication of the degree of homology. In someembodiments, two sequences are considered to be substantially homologousif at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more of their corresponding residues arehomologous over a relevant stretch of residues. In some embodiments, therelevant stretch is a complete sequence. In some embodiments, therelevant stretch is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300,325, 350, 375, 400, 425, 450, 475, 500 or more residues.

Substantial identity: The phrase “substantial identity” is used hereinto refer to a comparison between amino acid or nucleic acid sequences.As will be appreciated by those of ordinary skill in the art, twosequences are generally considered to be “substantially identical” ifthey contain identical residues in corresponding positions. As is wellknown in this art, amino acid or nucleic acid sequences may be comparedusing any of a variety of algorithms, including those available incommercial computer programs such as BLASTN for nucleotide sequences andBLASTP, gapped BLAST, and PSI-BLAST for amino acid sequences. Exemplarysuch programs are described in Altschul, et al., Basic local alignmentsearch tool, J. Mol. Biol., 215(3): 403-410, 1990; Altschul, et al.,Methods in Enzymology; Altschul et al., Nucleic Acids Res. 25:3389-3402,1997; Baxevanis et al., Bioinformatics: A Practical Guide to theAnalysis of Genes and Proteins, Wiley, 1998; and Misener, et al.,(eds.), Bioinformatics Methods and Protocols (Methods in MolecularBiology, Vol. 132) Humana Press, 1999. In addition to identifyingidentical sequences, the programs mentioned above typically provide anindication of the degree of identity. In some embodiments, two sequencesare considered to be substantially identical if at least 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more of their corresponding residues are identical over arelevant stretch of residues. In some embodiments, the relevant stretchis a complete sequence. In some embodiments, the relevant stretch is atleast 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400,425, 450, 475, 500 or more residues.

Target tissues: As used herein, the term “target tissues” refers to anytissue that is affected by the lysosomal storage disease to be treatedor any tissue in which the deficient lysosomal enzyme is normallyexpressed. In some embodiments, target tissues include those tissues inwhich there is a detectable or abnormally high amount of enzymesubstrate, for example stored in the cellular lysosomes of the tissue,in patients suffering from or susceptible to the lysosomal storagedisease. In some embodiments, target tissues include those tissues thatdisplay disease-associated pathology, symptom, or feature. In someembodiments, target tissues include those tissues in which the deficientlysosomal enzyme is normally expressed at an elevated level. As usedherein, a target tissue may be a brain target tissue, a spinal cordtarget tissue an/or a peripheral target tissue. Exemplary target tissuesare described in detail below.

Therapeutic moiety: As used herein, the term “therapeutic moiety” refersto a portion of a molecule that renders the therapeutic effect of themolecule. In some embodiments, a therapeutic moiety is a polypeptidehaving therapeutic activity.

Therapeutically effective amount: As used herein, the term“therapeutically effective amount” refers to an amount of a therapeuticprotein (e.g., replacement enzyme) which confers a therapeutic effect onthe treated subject, at a reasonable benefit/risk ratio applicable toany medical treatment. The therapeutic effect may be objective (i.e.,measurable by some test or marker) or subjective (i.e., subject gives anindication of or feels an effect). In particular, the “therapeuticallyeffective amount” refers to an amount of a therapeutic protein orcomposition effective to treat, ameliorate, or prevent a desired diseaseor condition, or to exhibit a detectable therapeutic or preventativeeffect, such as by ameliorating symptoms associated with the disease,preventing or delaying the onset of the disease, and/or also lesseningthe severity or frequency of symptoms of the disease. A therapeuticallyeffective amount is commonly administered in a dosing regimen that maycomprise multiple unit doses. For any particular therapeutic protein, atherapeutically effective amount (and/or an appropriate unit dose withinan effective dosing regimen) may vary, for example, depending on routeof administration, on combination with other pharmaceutical agents.Also, the specific therapeutically effective amount (and/or unit dose)for any particular patient may depend upon a variety of factorsincluding the disorder being treated and the severity of the disorder;the activity of the specific pharmaceutical agent employed; the specificcomposition employed; the age, body weight, general health, sex and dietof the patient; the time of administration, route of administration,and/or rate of excretion or metabolism of the specific fusion proteinemployed; the duration of the treatment: and like factors as is wellknown in the medical arts.

Tolerable: As used herein, the terms “tolerable” and “tolerability”refer to the ability of the pharmaceutical compositions of the presentinvention to not elicit an adverse reaction in the subject to whom suchcomposition is administered, or alternatively not to elicit a seriousadverse reaction in the subject to whom such composition isadministered. In some embodiments, the pharmaceutical compositions ofthe present invention are well tolerated by the subject to whom suchcompositions is administered.

Treatment: As used herein, the term “treatment” (also “treat” or“treating”) refers to any administration of a therapeutic protein (e.g.,lysosomal enzyme) that partially or completely alleviates, ameliorates,relieves, inhibits, delays onset of, reduces severity of and/or reducesincidence of one or more symptoms or features of a particular disease,disorder, and/or condition (e.g., Hunters syndrome). Such treatment maybe of a subject who does not exhibit signs of the relevant disease,disorder and/or condition and/or of a subject who exhibits only earlysigns of the disease, disorder, and/or condition. Alternatively oradditionally, such treatment may be of a subject who exhibits one ormore established signs of the relevant disease, disorder and/orcondition.

DETAILED DESCRIPTION

The present invention provides, among other things, an effective methodfor treating Hunter syndrome, in particular, Hunter syndrome withcognitive impairment based on intrathecal administration of recombinantiduronate-2-sulfatase (I2S) enzyme. In some embodiments, the presentinvention provides a method of treating Hunter syndrome by administeringintrathecally to a subject in need of treatment a recombinantiduronate-2-sulfatase (I2S) enzyme at a therapeutically effective doseand an administration interval for a treatment period sufficient toimprove, stabilize or reduce declining of one or more cognitive,adaptive, motor, and/or executive functions relative to a control (e.g.,baseline pre-treatment assessment or measurement) and/or to decreaseglycosaminoglycan (GAG) level in the cerebrospinal fluid (CSF) relativeto a control (e.g., baseline pre-treatment assessment or measurement).In some embodiments, the intrathecal administration is performed inconjunction with intravenous administration.

Various aspects of the invention are described in detail in thefollowing sections. The use of sections is not meant to limit theinvention. Each section can apply to any aspect of the invention. Inthis application, the use of “or” means “and/or” unless statedotherwise.

Recombinant Iduronate-2-sulfatase (I2S) Enzyme

As used herein, the term “recombinant iduronate-2-sulfatase (I2S)enzyme” encompasses any molecule or a portion of a molecule that cansubstitute for naturally-occurring Iduronate-2-sulfatase (I2S) enzymeactivity or rescue one or more phenotypes or symptoms associated withI2S-deficiency. In some embodiments, a recombinant I2S enzyme suitablefor the invention is a polypeptide having an N-terminus and a C-terminusand an amino acid sequence substantially similar or identical to maturehuman I2S protein. The terms “protein” and “enzyme” are usedinter-changeably in connection with I2S. A recombinant enzyme or proteinis also referred to as replacement enzyme or protein in thisapplication.

Typically, the human I2S protein is produced as a precursor form. Theprecursor form of human I2S contains a signal peptide (amino acidresidues 1-25 of the full length precursor), a pro-peptide (amino acidresidues 26-33 of the full length precursor), and a chain (residues34-550 of the full length precursor) that may be further processed intothe 42 kDa chain (residues 34-455 of the full length precursor) and the14 kDa chain (residues 446-550 of the full length precursor). Typically,the precursor form is also referred to as full-length precursor orfull-length I2S protein, which contains 550 amino acids. The amino acidsequences of the mature form (SEQ ID NO:1) having the signal peptideremoved and full-length precursor (SEQ ID NO:2) of a typical wild-typeor naturally-occurring human I2S protein are shown in Table 1.

TABLE 1  Human Iduronate-2-sulfatase Mature FormSETQANSTTDALNVLLIIVDDLRPSLGCYGDKLVRSPNIDQLASHSLLFQNAFAQQAVCAPSRVSFLTGRRPDTTRLYDFVSYWRVHAGNFSTIPQYFKENGYVTMSVGKVFHPGISSNHTDDSPYSWSFPPYHPSSEKYENTKTCRGPDGELHANLLCPVDVLDVPEGTLPDKQSTEQAIQLLEKMKTSASPFFLAVGYHKPHIPFRYPREFQKLYPLENITLAPDPEVPDGLPPVAYNPWMDIRQREDVQALNISVPYGPIPVDFQRKIRQSYFASVSYLDTQVGRLLSALDDLQLANSTIIAFTSDHGWALGEHGEWAKYSNFDVATHVPLIFYVPGRTASLPEAGEKLFPYLDPFDSASQLMEPGRQSMDLVELVSLFPTLAGLAGLQVPPRCPVPSFHVELCREGKNLLKHFRFRDLEEDPYLPGNPRELIAYSQYPRPSDIPQWNSDKPSLADIKIMGYSIRTIDYRYTVWVGFNPDEFLANFSDIHAGELYFVDSDPLQDHNMYNDSQGGD LFQLLMP (SEQ ID NO: 1) Full-LengthMPPPRTGRGLLWLGLVLSSVCVALGSETQANSTTDAL PrecursorNVLLIIVDDLRPSLGCYGDKLVRSPNIDQLASHSLLFQNAFAQQAVCAPSRVSFLTGRRPDTTRLYDFNSYWRVHAGNFSTIPQYFKENGYVTMSVGKVFHPGISSNHTDDSPYSWSFPPYHPSSEKYENTKTCRGPDGELHANLLCPVDVLDVPEGTLPDKQSTEQAIQLLEKMKTSASPFFLAVGYHKPHIPFRYPKEFQKLYPLENITLAPDPEVPDGLPPVAYNPWMDIRQREDVQALNISVPYGPIPVDFQRKIRQSYFASVSYLDTQVGRLLSALDDLQLANSTIIAFTSDHGWALGEHGEWAKYSNFDVATHVPLIFYVPGRTASLPEAGEKLFPYLDPFDSASQLMEPGRQSMDLVELVELFPTLAGLAGLQVPPRCPVPSFHVELCREGKNLLKHFRFRDLEEDPYLPGNPRELIAYSQYPRPSDIPQWNSDKPSLKDIKIMGYSIRTIDYRYTVWVGFNPDEFLANFSDIHAGELYFVDSDPLQDHWMYNDSQGGDLFQLLMF(SEQ  ID NO: 2)

Thus, in some embodiments, a recombinant I2S enzyme suitable for thepresent invention is mature human I2S protein (SEQ ID NO: 1). In someembodiments, a suitable recombinant I2S enzyme may be a homologue or ananalogue of mature human I2S protein. For example, a homologue or ananalogue of mature human I2S protein may be a modified mature human I2Sprotein containing one or more amino acid substitutions, deletions,and/or insertions as compared to a wild-type or naturally-occurring I2Sprotein (e.g., SEQ ID NO: 1), while retaining substantial I2S proteinactivity. Thus, in some embodiments, a recombinant I2S enzyme suitablefor the present invention is substantially homologous to mature humanI2S protein (SEQ ID NO:1). In some embodiments, a recombinant I2S enzymesuitable for the present invention has an amino acid sequence at least50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more homologous to SEQ ID NO: 1. In someembodiments, a recombinant I2S enzyme suitable for the present inventionis substantially identical to mature human I2S protein (SEQ ID NO:1). Insome embodiments, a recombinant I2S enzyme suitable for the presentinvention has an amino acid sequence at least 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moreidentical to SEQ ID NO:1. In some embodiments, a recombinant I2S enzymesuitable for the present invention contains a fragment or a portion ofmature human I2S protein.

Alternatively, a recombinant I2S enzyme suitable for the presentinvention is ill-length 128 protein. In some embodiments, a suitablerecombinant I2S enzyme may be a homologue or an analogue of full-lengthhuman I2S protein. For example, a homologue or an analogue offull-length human I2S protein may be a modified full-length human I2Sprotein containing one or more amino acid substitutions, deletions,and/or insertions as compared to a wild-type or naturally-occurringfull-length I2S protein (e.g., SEQ ID NO:2), while retaining substantialI2S protein activity, Thus, In some embodiments, a recombinant I2Senzyme suitable for the present invention is substantially homologous tofull-length human 128 protein (SEQ ID NO:2). In some embodiments, arecombinant I2S enzyme suitable for the present invention has an aminoacid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous to SEQ IDNO:2. In some embodiments, a recombinant I2S enzyme suitable for thepresent invention is substantially identical to SEQ ID NO:2. In someembodiments, a recombinant I2S enzyme suitable for the present inventionhas an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%,85% 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identicalto SEQ ID NO:2. In some embodiments, a recombinant I2S enzyme suitablefor the present invention contains a fragment or a portion offull-length human I2S protein. As used herein, a fill-length I2S proteintypically contains signal peptide sequence.

A recombinant I2S enzyme suitable for the present invention may beproduced by any available means. For example, replacement enzymes may berecombinantly produced by utilizing a host cell system engineered toexpress a replacement enzyme-encoding nucleic acid. Alternatively oradditionally, recombinant I2S enzymes may be produced by activatingendogenous genes. Alternatively or additionally, recombinant I2S enzymesmay be partially or fully prepared by chemical synthesis. Alternativelyor additionally, recombinant I2S enzymes may also be purified fromnatural sources.

Where enzymes are recombinantly produced, any expression system can beused. To give but a few examples, known expression systems include, forexample, egg, baculovirus, plant, yeast, or mammalian cells

In some embodiments, enzymes suitable for the present invention areproduced in mammalian cells. Non-limiting examples of mammalian cellsthat may be used in accordance with the present invention include BALB/cmouse myeloma line (NSO/1, ECACC No: 85110503); human retinoblasts(PER.C6, CruCell, Leiden, The Netherlands); monkey kidney CV1 linetransformed by SV40 (COS-7, ATCC CRL, 1651); human embryonic kidney line(293 or 293 cells subcloned for growth in suspension culture, Graham etal., J. Gen Virol., 36:59, 1977); human fibrosarcoma cell line (e.g.,HT1080); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamsterovary cells +/−DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA,77:4216, 1980); mouse sertoli cells (TM4, Mather, Biol. Reprod.,23:243-251, 1980); monkey kidney cells (CV1 ATCC CCL 70); African greenmonkey kidney cells (VERO-76, ATCC CRL-1 587); human cervical carcinomacells (HeLa, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34);buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138,ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor(MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad.Sci., 383:44-68, 1982); MRC 5 cells; FS4 cells; and a human hepatomaline (Hep G2).

In some embodiments, recombinant I2S enzymes suitable for the presentinvention are produced from human cells. In some embodiments,recombinant I2S enzymes suitable for the present invention are producedfrom CHO cells.

In some embodiments, recombinant I2S enzymes suitable for the presentinvention contain a moiety that binds to a receptor on the surface ofbrain cells to facilitate cellular uptake and/or lysosomal targeting.For example, such a receptor may be the cation-independentmannose-6-phosphate receptor (CI-MPR) which binds themannose-6-phosphate (M6P) residues. In addition, the CI-MPR also bindsother proteins including IGF-II. In some embodiments, a recombinant I2Senzyme suitable for the present invention contains M6P residues on thesurface of the protein. In some embodiments, a recombinant I2S enzymesuitable for the present invention may contain bis-phosphorylatedoligosaccharides which have higher binding affinity to the CI-MPR. Insome embodiments, a suitable recombinant I2S enzyme contains up to aboutan average of about at least 20% bis-phosphorylated oligosaccharides perenzyme. In other embodiments, a suitable enzyme may contain about 10%,15%, 18%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% bis-phosphorylatedoligosaccharides per enzyme. While such his-phosphorylatedoligosaccharides may be naturally present on the enzyme, it should benoted that the enzymes may be modified to possess such oligosaccharides.For example, suitable recombinant I2S enzymes may be modified by certainenzymes which are capable of catalyzing the transfer ofN-acetylglucosamine-L-phosphate from UDP-GlcNAc to the 6′ position ofα-1,2-linked mannoses on lysosomal enzymes. Methods and compositions forproducing and using such enzymes are described by, for example, Canfieldet al. in U.S. Pat. No. 6,537,785, and U.S. Pat. No. 6,534,300, eachincorporated herein by reference.

In some embodiments, recombinant I2S enzymes for use in the presentinvention may be conjugated or fused to a lysosomal targeting moietythat is capable of binding to a receptor on the surface of brain cells.A suitable lysosomal targeting moiety can be IGF-I, IGF-II, RAP, p97,and variants, homologues or fragments thereof (e.g., including thosepeptide having a sequence at least 70%, 75%, 80%, 85%, 90%, or 95%identical to a wild-type mature human IGF-I, IGF-II, RAP, p97 peptidesequence),

In some embodiments, recombinant I2S enzymes suitable for the presentinvention have not been modified to enhance delivery or transport ofsuch agents across the BBB and into the CNS.

Intrathecal Administration

In some embodiments, a recombinant I2S enzyme is delivered to the CNS byadministering into the cerebrospinal fluid (CSF) of a subject in need oftreatment. In some embodiments, intrathecal administration is used todeliver a desired replacement enzyme into the CSF. As used herein,intrathecal administration (also referred to as intrathecal injection)refers to an injection into the spinal canal (intrathecal spacesurrounding the spinal cord). Various techniques may be used including,without limitation, lateral cerebroventricular injection through aburrhole or cisternal or lumbar puncture or the like. Exemplary methodsare described in International Application WO2011/163648, entitled “CNSDelivery of Therapeutic Agents”, the contents of which are incorporatedherein by reference.

According to the present invention, a recombinant I2S enzyme may beinjected at any region surrounding the spinal canal. In someembodiments, an enzyme is injected into the lumbar area or the cisternamagna or intraventricularly into a cerebral ventricle space. As usedherein, the term “lumbar region” or “lumbar area” refers to the areabetween the third and fourth lumbar (lower back) vertebrae and, moreinclusively, the L2-S1 region of the spine. Typically, intrathecalinjection via the lumbar region or lumber area is also referred to as“lumbar IT delivery” or “lumbar IT administration.” the term “cisternamagna” refers to the space around and below the cerebellum via theopening between the skull and the top of the spine. Typically,intrathecal injection via cisterna magna is also referred to as“cisterna magna delivery.” The term “cerebral ventricle” refers to thecavities in the brain that are continuous with the central canal of thespinal cord. Typically, injections via the cerebral ventricle cavitiesare referred to as intraventricular Cerebral (ICV) delivery.

In some embodiments, “intrathecal administration” or “intrathecaldelivery” according to the present invention refers to lumbar ITadministration or delivery, for example, delivered between the third andfourth lumbar (lower back) vertebrae and, more inclusively, the L2-S1region of the spine. It is contemplated that lumbar IT administration ordelivery distinguishes over cisterna magna delivery in that lumbar ITadministration or delivery according to our invention provides betterand more effective delivery to the distal spinal canal, while cisternamagna delivery, among other things, typically does not deliver well tothe distal spinal canal. In some embodiments, intrathecal administrationis performed in the L5-L6, L4-L5, L3-L4, L2-L3, and/or L2-S1 regions ofthe spine.

Formulations for IT Delivery

In some embodiments, a desired amount of recombinant I2S enzyme isdelivered in a formulation suitable for intrathecal delivery.Particularly useful formulations are capable of solubilizing highconcentrations of recombinant I2S enzyme and are further characterizedby improved stability and improved tolerability when administeredintrathecally to the CNS of a subject in need thereof. As used herein,the term “soluble” as it relates to a recombinant I2S enzyme refers tothe ability of the recombinant I2S enzyme to form a homogenous solution.

Thus, suitable formulations for intrathecal administration may contain arecombinant I2S enzyme at various concentrations. In some embodiments,suitable formulations may contain a recombinant I2S enzyme at aconcentration up to about 300 mg/ml (e.g., up to about 250 mg/ml, up toabout 200 mg/ml, up to about 150 mg/ml, up to about 100 mg/ml, up toabout 90 mg/ml, up to about 80 mg/ml, up to about 70 mg/ml, up to about60 mg/ml, up to about 50 mg/ml, up to about 40 mg/ml, up to about 30mg/ml, up to about 25 mg/ml, up to about 20 mg/ml, up to about 10mg/ml). In some embodiments, suitable formulations may contain arecombinant I2S enzyme at a concentration ranging between about 0-300mg/ml (e.g., about 1-250 mg/ml, about 1-200 mg/ml, about 1-150 mg/ml,about 1-100 mg/ml, about 10-100 mg/ml, about 10-80 mg/ml, about 10-70mg/ml, about 1-60 mg/ml, about 1-50 mg/ml, about 10-150 mg/ml, about1-30 mg/ml). In some embodiments, formulations suitable for intrathecaldelivery may contain a recombinant I2S enzyme at a concentration ofapproximately 1 mg/ml, 3 mg/ml, 5 mg/ml, 10 mg/ml, 15 mg/ml, 20 mg/ml,25 mg/ml, 50 mg/ml, 75 mg/ml, 100 mg/ml, 150 mg/ml, 200 mg/ml, 250 mg/mlor 300 mg/ml.

In some embodiments, isotonic solutions are used. In some embodiments,slightly hypertonic solutions (e.g., up to 300 mM (e.g., up to 250 mM,200 mM, 175 mM, 150 mM, 125 mM) sodium chloride in 5 mM sodium phosphateat pH 7.0) and sugar-containing solutions (e.g., up to 3% (e.g., up to2.4%, 2.0%, 1.5%, 1.0%) sucrose in 5 mM sodium phosphate at pH 7.0) havebeen demonstrated to be well tolerated in monkeys. In some embodiments,a suitable CNS bolus formulation composition is saline (e.g., I50 mMNaCl in water).

As non-limiting examples, Table 2 below list exemplary pH and excipientssuitable for maintaining the solubility and stability of a recombinantI2S in a formulation for intrathecal administration.

TABLE 2 Exemplary pH and excipients Parameter Typical Range/TypeRationale pH 4 to 8.0 For stability Sometimes also for solubility Buffertype acetate, succinate, citrate, To maintain optimal pH histidine,phosphate or Tris May also affect stability Buffer 5-50 mM To maintainpH concentration May also stabilize or add ionic strength TonicifierNaCl, sugars, mannitol To render iso-osmotic or isotonic solutionsSurfactant Polysorbate 20, polysorbate 80 To stabilize againstinterfaces and shear Other Amino acids (e.g. arginine) at For enhancedsolubility or stability tens to hundreds of mM

In some embodiments, formulations suitable for the present inventioncontain an amount of buffer sufficient to maintain the optimal pH ofsaid formulation between about 4.0-8.0, between about 5.0-7.5, betweenabout 5.5-7.0, between about 6.0-7.0 and between about 6.0-7.5. Suitablebuffers include, for example acetate, succinate, citrate, phosphate,other organic acids and tris(hydroxymethyl)aminomethane (“Tris”).Suitable buffer concentrations can be from about 1 mM to about 100 mM,from about 1 mM to about 50 mM, or from about 3 mM to about 20 mM,depending, for example, on the buffer and the desired isotonicity of theformulation. In some embodiments, a suitable buffering agent is presentat a concentration of approximately 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75mM, 80 mM, 85 mM, 90 mM, 95 mM, or 100 mM. In particular embodiments, aformulation suitable for the present invention contains less than about50 mM (e.g., less than about 45 mM, 40 mM, 35 mM, 30 mM, 25 mM, 20 mM,15 mM, 10 mM, or 5 mM) of phosphate (e.g., sodium phosphate).

In some embodiments, formulations contain an isotonicity agent to keepthe formulations isotonic. As used in connection with IT delivery, by“isotonic” is meant that the formulation of interest has essentially thesame osmolarity as human CSF. Isotonic formulations will generally havean osmolarity from about 240 mOsm/kg to about 350 mOsm/kg. Isotonicitycan be measured using, for example, a vapor pressure or freezing pointtype osmometers. Exemplary isotonicity agents include, but are notlimited to, glycine, sorbitol, mannitol, sodium chloride and arginine.In some embodiments, suitable isotonic agents may be present informulations at a concentration from about 0.01-5% (e.g., 0.05, 0.1,0.15, 02, 0.3, 0, 0.5, 0.75, 1.0, 1.25, 1.5, 2.0, 2.5, 3.0, 4.0 or 5.0%)by weight.

In some embodiments, formulations may contain a stabilizing agent toprotect the protein. Typically, a suitable stabilizing agent is anon-reducing sugar such as sucrose, raffinose, trehalose, or amino acidssuch as glycine, arginine and methionine. The amount of stabilizingagent in a formulation is generally such that the formulation will beisotonic. However, hypertonic formulations may also be suitable. Inaddition, the amount of stabilizing agent must not be too low such thatan unacceptable amount of degradation/aggregation of the therapeuticagent occurs. Exemplary stabilizing agent concentrations in theformulation may range from about 1 mM to about 400 mM (e.g., from about30 mM to about 300 mM, and from about 50 mM to about 100 mM), oralternatively, from 0.1% to 15% (e.g., from 1% to 10%, from 5% to 15%,from 5% to 10%) by weight. In some embodiments, the ratio of the massamount of the stabilizing agent and the therapeutic agent is about 1:1.In other embodiments, the ratio of the mass amount of the stabilizingagent and the therapeutic agent can be about 0.1:1, 0.2:1, 0.25:1,0.4:1, 0.5:1, 1:1, 2:1, 2.6:1, 3:1, 4:1, 5:1, 10:1, or 20:1. In someembodiments, suitable for lyophilization, the stabilizing agent is alsoa lyoprotectant.

In some embodiments, it is desirable to add a surfactant toformulations. Exemplary surfactants include nonionic surfactants such asPolysorbates (e.g., Polysorbates 20 or 80); poloxamers (e.g., poloxamer188); Triton; sodium dodecyl sulfate (SDS); sodium laurel sulfate;sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, orstearyl-sulfobetaine; lauryl-, myristyl-, linoleyl- orstearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine;lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-,myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine(e.g., lauroamidopropyl); myristarnidopropyl-, palmidopropyl, orisostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodiummethyl ofeyl-taurate; and the MONAQUAT™ series (Mona Industries, Inc.,Paterson, N.J.), polymethyl glycol, polypropyl glycol, and copolymers ofethylene and propylene glycol (e.g., Pluronics, PF68, etc). Typically,the amount of surfactant added is such that it reduces aggregation ofthe protein and minimizes the formation of particulates oreffervescences. For example, a surfactant may be present in aformulation at a concentration from about 0.001-0.5% (e.g., about0.001-0.04%, about 0.001-0.03%, about 0.001-0.02%, about 0.001-0.01%,about 0.001-0.008%, about 0.001-0.006%, about 0.001-0.004%, about0.005-0.05%, or 0.005-0.01%). In particular, a surfactant may be presentin a formulation at a concentration of approximately 0.001%, 0.002%,0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%,0.03%, 0.04%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, or 0.5%, etc.

In some embodiments, suitable formulations may further include one ormore bulking agents, in particular, for lyophilized formylations. A“bulking agent” is a compound which adds mass to the lyophilized mixtureand contributes to the physical structure of the lyophilized cake. Forexample, a bulking agent may improve the appearance of lyophilized cake(e.g., essentially uniform lyophilized cake). Suitable bulking agentsinclude, but are not limited to, sodium chloride, lactose, mannitol,glycine, sucrose, trehalose, hydroxyethyl starch. Exemplaryconcentrations of bulking agents are from about 1% to about 10% (e.g.,1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 5.5%, 6.0%, 6.5%,7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9.5%, and 10.0%).

Formulations suitable for the present invention can be assessed based onproduct quality analysis, reconstitution time (if lyophilized), qualityof reconstitution (if lyophilized), high molecular weight, moisture, andglass transition temperature. Typically, protein quality and productanalysis include product degradation rate analysis using methodsincluding, but not limited to, size exclusion HPLC (SE-HPLC), cationexchange-HPLC (CEX-HPLC), X-ray diffraction (XRD), modulateddifferential scanning calorimetry (mDSC), reversed phase HPLC (RP-HPLC),multi-angle light scattering (MALS), fluorescence, ultravioletabsorption, nephelometry, capillary electrophoresis (CE), SDS-PAGE, andcombinations thereof. In some embodiments, evaluation of product inaccordance with the present invention may include a step of evaluatingappearance (either liquid or cake appearance).

Generally, formulations (lyophilized or aqueous) can be stored forextended periods of time at room temperature. Storage temperature maytypically range from 0° C. to 45° C. (e.g., 4° C., 20° C., 25° C., 45°C. etc.). Formulations may be stored for a period of months to a periodof years. Storage time generally will be 24 months, 12 months, 6 months,4.5 months, 3 months, 2 months or 1 month. Formulations can be storeddirectly in the container used for administration, eliminating transfersteps.

Formulations can be stored directly in the lyophilization container (iflyophilized), which may also function as the reconstitution vessel,eliminating transfer steps. Alternatively, lyophilized productformulations may be measured into smaller increments for storage.Storage should generally avoid circumstances that lead to degradation ofthe proteins, including but not limited to exposure to sunlight, UVradiation, other forms of electromagnetic radiation, excessive heat orcold, rapid thermal shock, and mechanical shock.

In some embodiments, formulations suitable for the present invention arein a liquid or aqueous form. In some embodiments, formulations for thepresent invention are lyophilized. Such lyophilized formulations may bereconstituted by adding one or more diluents thereto prior toadministration to a subject. Suitable diluents include, but are notlimited to, sterile water, bacteriostatic water for injection andsterile saline solution preferably, upon reconstitution, the therapeuticagent contained therein is stable, soluble and demonstrates tolerabilityupon administration to a subject

Suitable formulations are characterized by their tolerability. As usedherein, the terms “tolerable” and “tolerability” refer to the ability ofa formulation to not elicit an adverse reaction, in particular, not toelicit a serious adverse reaction in the subject to whom suchformulation is administered. In some embodiments, a formulationparticularly useful for the present invention is well tolerated by thesubject to whom such formulation is administered.

Additional exemplary formulations suitable for intrathecal delivery of arecombinant I2S enzyme are described in International Application WOWO2011/163649, entitled “METHODS AND COMPOSITIONS FOR CNS DELIVERY OFIDURONATE-2-SULFATASE,” the contents of which are hereby incorporated byreference.

Dosing Regimen

Typically, a therapeutically effective amount of a recombinant I2S isadministered in a dosing regimen that may comprise multiple unit doses.A dosing regimen suitable for any particular patient may depend upon avariety of factors including the disorder being treated and the severityof the disorder; the age, body weight, general health, sex and diet ofthe patient; the time of administration, and/or rate of excretion ormetabolism; the duration of the treatment; and like factors as is wellknown in the medical arts.

Unit dose used in a dosing regimen is also referred to as atherapeutically effective dose. A therapeutically effective dose may bedefined in various ways. For example, a therapeutically effective dosemay be defined by the total amount of recombinant I2S enzymeadministered at each time. Thus, in some embodiments, a therapeuticallyeffective dose according to the invention as or greater than about 1 mg,5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg or 100 mg perdose. In particular embodiments, a therapeutically effective dose is orgreater than about 10 mg per dose. In particular embodiments, atherapeutically effective dose is or greater than about 30 mg per dose.In some embodiments, a therapeutically effective dose is less than about50 mg, 45 mg, 40 mg, 35 mg, 30 mg, 25 mg, 20 mg, 15 mg, or 10 mg perdose. In particular embodiments, a therapeutically effective dose isless than about 30 mg per dose. In some embodiments, a therapeuticallyeffective dose ranges between about 1-100 mg, about 5-100 mg, about 5-90mg, about 5-80 mg, about 5-70 mg, about 5-60 mg, about 5-60 mg, about10-100 mg, about 10-90 mg, about 10-80 mg, about 10-70 mg, about 10-60mg, or about 10-50 mg.

Alternatively, a therapeutically effective dose may be defined by theamount of recombinant I2S enzyme administered relative to the brainweight. In some embodiments, a therapeutically effective dose accordingto the present invention ranges from about 0.005 mg/kg brain weight to500 mg/kg brain weight, e.g., from about 0.005 mg/kg brain weight to 400mg/kg brain weight, from about 0.005 mg/kg brain weight to 300 mg/kgbrain weight, from about 0.005 mg/kg brain weight to 200 mg/kg brainweight, from about 0.005 mg/kg brain weight to 100 mg/kg brain weight,from about 0.005 mg/kg brain weight to 90 mg/kg brain weight, from about0.005 mg/kg brain weight to 80 mg/kg brain weight, from about 0.005mg/kg brain weight to 70 mg/kg brain weight, from about 0.005 mg/kgbrain weight to 60 mg/kg brain weight, from about 0.005 mg/kg brainweight to 50 mg/kg brain weight, from about 0.005 mg/kg brain weight to40 mg/kg brain weight, from about 0.005 mg/kg brain weight to 30 mg/kgbrain weight, from about 0.005 mg/kg brain weight to 25 mg/kg brainweight, from about 0.005 mg/kg brain weight to 20 mg/kg brain weight,from about 0.005 mg/kg brain weight to 15 mg/kg brain weight, from about0.005 mg/kg brain weight to 10 mg/kg brain weight.

In some embodiments, the therapeutically effective dose is or greaterthan about 0.1 mg/kg brain weight, about 0.5 mg/kg brain weight, about1.0 mg/kg brain weight, about 3 mg/kg brain weight, about 5 mg/kg brainweight, about 10 mg/kg brain weight, about 15 mg/kg brain weight, about20 mg/kg brain weight, about 30 mg/kg brain weight, about 40 mg/kg brainweight, about 50 mg/kg brain weight, about 60 mg/kg brain weight, about70 mg/kg brain weight, about 80 mg/kg brain weight, about 90 mg/kg brainweight, about 100 mg/kg brain weight, about 150 mg/kg brain weight,about 200 mg/kg brain weight, about 250 mg/kg brain weight, about 300mg/kg brain weight, about 350 mg/kg brain weight, about 400 mg/kg brainweight, about 450 mg/kg brain weight, or about 500 mg/kg brain weight.

In some embodiments, the therapeutically effective dose may also beadjusted by age or body weight, especially in children under the age of3. As one skilled in the art would appreciate, brain weights changerapidly during the first 3 years of life, reaching a plateau thereafterand body weights can be correlated in young children. See, Dekaban AS.“Changes in brain weights during the span of human life: relation ofbrain weights to body heights and body weights,” Ann Neural 1978;4:345-56. Therefore, children younger than 3 years may require anadjusted (typically smaller) dose compared to older children and adults.In some embodiments, dosages used in young children may be adjustedaccording to the guide to the adjustment of dose based on brain weightin young children provided below (see Table 3).

TABLE 3 Change in Brain Wight During Early Human Development Body Weight(kg) Age No. of Brain Weight (kg) Body Height (m) % Group Age (yr)Brains Mean SD SEM % Change^(a) Mean SD SEM % Change^(a) Mean SD SEMChange^(a) 1 NB (0-10 d) 241 0.38 0.09 0.00 . . . 0.50 0.05 0.00 . . .2.95 0.47 0.03 . . . 2 0.5 (4-8 mo) 87 0.64 0.16 0.01 66.8 0.59 0.090.01 18.6 5.88 3.06 0.32 99.4 3 1 (9-18 mo) 33 0.97 0.16 0.02 50.6 0.760.11 0.02 28.5 9.47 2.57 0.41 61.2 4 2 (19-30 mo) 55 1.12 0.20 0.02 16.20.85 0.12 0.01 11.7 13.20 3.57 0.49 39.3 5 3 (31-43 mo) 19 1.27 0.210.04 12.8 0.94 0.09 0.02 11.0 15.55 3.43 0.78 17.9 6 4-5 29 1.50 0.020.00 2.3 1.06 0.03 0.00 12.5 19.46 1.21 0.22 25.1

In some embodiments, the therapeutically effective dose may also bedefined by mg/15 cc of CSF. As one skilled in the art would appreciate,therapeutically effective doses based on brain weights and body weightscan be converted to mg/I5 cc of CSF. For example, the volume of CSF inadult humans is approximately 150 mL (Johanson C E, et al. “Multiplicityof cerebrospinal fluid functions: New challenges in health and disease,”Cerebrospinal Fluid Res, 2008 May 14; 5:10). Therefore, single doseinjections of 0.1 mg to 50 mg protein to adults would be approximately0.01 mg/15 cc of CSF (0.1 mg) to 5.0 mg/15 cc of CSF (50 mg) doses inadults.

Recombinant I2S enzymes can be administered at regular intervals. Insome embodiments, a therapeutically effective dose may be administeredintrathecally periodically at regular intervals, e.g., once every year,once every six months (or twice a year), once every five months, onceevery four months, once every three months, bimonthly (once every twomonths), monthly (once every month or once every four weeks), once everythree weeks, biweekly (once every two weeks), weekly (once every week),or at a variable interval.

Intrathecal administration may be performed in conjunction withintravenous administration of a recombinant I2S enzyme. In someembodiments, intravenous administration of a recombinant I2S enzyme isweekly. In some embodiments, the intravenous administration of arecombinant I2S enzyme is weekly except the week when the intrathecaladministration is performed. In some embodiments, intravenousadministration of a recombinant I2S enzyme is biweekly, once every threeweeks, monthly (once every four weeks), twice a month, once every two,three, four, five, or six months. In some embodiments, intravenousadministration of a recombinant I2S enzyme is at a dose of about 0.1,0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 09, or 1.0 mg/kg body weight. In someembodiments, the intravenous administration of the recombinant I2Senzyme is at a dose of about 0.5 mg/kg body weight.

Device for Intrathecal Delivery

Various devices may be used for intrathecal delivery according to thepresent invention. In some embodiments, a device for intrathecaladministration contains a fluid access port (e.g., injectable port); ahollow body (e.g., catheter) having a first flow orifice in fluidcommunication with the fluid access port and a second flow orificeconfigured for insertion into spinal cord; and a securing mechanism forsecuring the insertion of the hollow body in the spinal cord. As anon-limiting example, a suitable securing mechanism contains one or morenobs mounted on the surface of the hollow body and a sutured ringadjustable over the one or more nobs to prevent the hollow body (e.g.,catheter) from slipping out of the spinal cord. In various embodiments,the fluid access port comprises a reservoir. In some embodiments, thefluid access port comprises a mechanical pump (e.g., an infusion pump).In some embodiments, an implanted catheter is connected to either areservoir (e.g., for bolus delivery), or an infusion pump. The fluidaccess port may be implanted or external

In some embodiments, intrathecal administration may be performed byeither lumbar puncture (i.e., slow bolus) or via a port-catheterdelivery system (i.e., infusion or bolus). In some embodiments, thecatheter is inserted between the laminae of the lumbar vertebrae and thetip is threaded up the thecal space to the desired level (generallyL3-L4).

Relative to intravenous administration, a single dose volume suitablefor intrathecal administration is typically small. Typically,intrathecal delivery according to the present invention maintains thebalance of the composition of the CSF as well as the intracranialpressure of the subject. In some embodiments, intrathecal delivery isperformed absent the corresponding removal of CSF from a subject. Insome embodiments, a suitable single dose volume may be e.g., less thanabout 10 ml, 8 ml, 6 ml, 5 ml, 4 ml, 3 ml, 2 ml, 1.5 ml, 1 ml, or 0.5ml. In some embodiments, a suitable single dose volume may be about0.5-5 ml, 0.5-4 ml, 0.5-3 ml, 0.5-2 ml, 0.5-1 ml, 1-3 ml, 1-5 ml, 1.5-3ml, 1-4 ml, or 0.5-1.5 ml. In some embodiments, intrathecal deliveryaccording to the present invention involves a step of removing a desiredamount of CSF first. In some embodiments, less than about 10 ml (e.g.,less than about 9 ml, 8 ml, 7 nil, 6 ml, 5 ml, 4 ml, 3 ml, 2 ml, 1 ml)of CSF is first removed before IT administration. In those cases, asuitable single dose volume may be e.g., more than about 3 ml, 4 ml, 5ml, 6 ml, 7 ml, 8 ml, 9 ml, 10 ml, 15 ml, or 20 ml.

Various other devices may be used to effect intrathecal administrationof a therapeutic composition. For example, formulations containingdesired enzymes may be given using an Ommaya reservoir which is incommon use for intrathecally administering drugs for meningealcarcinomatosis (Lancet 2: 983-84, 1963). More specifically, in thismethod, a ventricular tube is inserted through a hole formed in theanterior horn and is connected to an Ommaya reservoir installed underthe scalp, and the reservoir is subcutaneously punctured tointrathecally deliver the particular enzyme being replaced, which isinjected into the reservoir. Other devices for intrathecaladministration of therapeutic compositions or formulations to anindividual are described in U.S. Pat. No. 6,217,552, incorporated hereinby reference. Alternatively, the drug may be intrathecally given, forexample, by a single injection, or continuous infusion. It should beunderstood that the dosage treatment may be in the form of a single doseadministration or multiple doses.

For injection, formulations of the invention can be formulated in liquidsolutions. In addition, the enzyme may be formulated in solid form andre-dissolved or suspended immediately prior to use. Lyophilized formsare also included. The injection can be, for example, in the form of abolus injection or continuous infusion (e.g., using infusion pumps) ofthe enzyme.

Typically, intrathecal administration can be through intermittent orcontinuous access to an implanted intrathecal drug delivery device(IDDD). In some embodiments, intrathecal administration is throughcontinuous access to an implanted IDDD for, e.g., greater than 0.1, 0.2,0.3, 0.4, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0 hours. Inother embodiments, intrathecal administration is through sustaineddelivery, e.g., “slow release” of a recombinant I2S enzyme, to a subjectfor at least one, two, three, four, five, six days, or one, two, three,four weeks or longer periods of time.

As used herein, the term “sustained delivery” refers to continualdelivery of a pharmaceutical formulation in vivo over a period of timefollowing administration for. e.g., at least several days, a week orseveral weeks. Sustained delivery of the composition can be demonstratedby, for example, the continued therapeutic effect of the enzyme overtime (e.g., sustained delivery of the enzyme can be demonstrated bycontinued reduced amount of storage granules in the subject).Alternatively, sustained delivery of the enzyme may be demonstrated bydetecting the presence of the enzyme in vive over time.

Immune Tolerance

Generally, intrathecal administration of a recombinant I2S enzymeaccording to the present invention does not result in severe adverseeffects in the subject. As used herein, severe adverse effects induce,but are not limited to, substantial immune response, toxicity, or death.As used herein, the term “substantial immune response” refers to severeor serious immune responses, such as adaptive T-cell immune responses.

Thus, in many embodiments, inventive methods according to the presentinvention do not involve concurrent immunosuppressant therapy (i.e., anyimmunosuppressant therapy used as pre-treatment/pre-conditioning or inparallel to the method). In some embodiments, inventive methodsaccording to the present invention do not involve an immune toleranceinduction in the subject being treated. In some embodiments, inventivemethods according to the present invention do not involve apre-treatment or preconditioning of the subject using T-cellimmunosuppressive agent.

In some embodiments intrathecal administration of therapeutic agents canmount an immune response against these agents. Thus, in someembodiments, it may be useful to render the subject receiving thereplacement enzyme tolerant to the enzyme replacement therapy. Immunetolerance may be induced using various methods known in the art. Forexample, an initial 30-60 day regimen of a T-cell immunosuppressiveagent such as cyclosporin A (CsA) and an antiproliferative agent, suchas, azathioprine (Aza), combined with weekly intrathecal infusions oflow doses of a desired replacement enzyme may be used.

Any immunosuppressant agent known to the skilled artisan may be employedtogether with a combination therapy of the invention. Suchimmunosuppressant agents include hut are not limited to cyclosporine,FK506, rapamycin, CTLA4-1g, and anti-TNF agents such as etanercept (seee.g. Moder, 2000, Ann. Allergy Asthma Immunol. 84, 280-284; Nevins,2000, Curr. Opin. Pediatr. 12, 146-150; Kurlberg et al., 2000, Scand. J.Immunol. 51, 224-230; Ideguchi et al., 2000, Neuroscience 95, 217-226;Potteret al., 1999, Arm. N.Y. Acad. Sci. 875, 159-174; Slavik et al.,1999, Immunol. Res. 19, 1-24; Gaziev et al., 1999, Bone MarrowTransplant, 25, 689-696; Henry, 1999, Clin. Transplant, 13, 209-220;Gummert et al., 1999, J. Am. Soc. Nephrol. 10, 1366-1380: Qi et al.,2000, Transplantation 69, 1275-1283). The anti-IL2 receptor(.alpha.-subunit) antibody daclizumab (e.g. Zenapax™), which has beendemonstrated effective in transplant patients, can also be used as animmunosuppressant agent (see e.g. Wiseman et al., 1999, Drugs 58,1029-1042. Beniaminovitz et al., 2000, N. Engl J. Med. 342, 613-619;Ponticelli et al., 1999, Drugs R. D. 1, 55-60; Berard et al., 1999,Pharmacotherapy 19, 1127-1137; Eckhoff et al., 2000, Transplantation 69,1867-1872; Ekberg et al, 2000, Transpl. Int. 13, 151-159). Additionalimmunosuppressant agents include but are not limited to anti-CD2 (Brancoet alt, 1999, Transplantation 68, 1588-1596: Przepiorka et al., 1998,Blood 92, 4066-4071), anti-CD4 (Marinova-Mutafchieva et al., 2000,Arthritis Rheum. 43, 638-644; Fishwild et al., 1999. Clin. Immunol. 92,138-152), and anti-CD40 ligand (Hong et al., 2000, Semin. Nephrol, 20,108-125; Chirmule et al., 2000, J. Virol, 74, 3345-3352; Ito et al.,2000, J. Immunol. 164, 1230-1235).

Pharmacokinetics, Pharmacodynamics, and Bioavailability

Among other things, intrathecally delivered recombinant I2S exhibitssuperior pharmacokinetics, pharmacodynamics and bioavailability in ahuman patient. Evaluation of I2S concentration-time profiles in CSF maybe evaluated directly by CSF sampling or indirectly by measuringsystemic serum I2S concentration-time profiles. Typically, however, dueto the limited number of clinically permissible CSF sample collections,I2S pharmacokinetics and pharmacodynamics profiles are evaluatedindirectly by sampling the blood periodically. The following standardabbreviations are used to represent the associated pharmacokineticparameters.

-   -   AUC_(inf) Area under the plasma concentration versus time curve        up to the last measurable concentration plus the AUC, calculated        using the linear trapezoidal rule from the zero time point to        the last quantifiable concentration and extrapolated from the        last measurable concentration (C_(last) at t_(last)) to        infinity: AUC_(INFobs)=AUC_(0-tlast)+C_(last)/Lambda z (where λz        is the first order rate constant associated with the terminal        (log-linear) portion of the curve)    -   AUC₀₋₁₂ Area under the curve between the time of dose and the 12        h time point    -   AUC₀₋₂₄ Area under the curve between the time of dose and the 24        h time point    -   AUC_(ss) Exposure at steady state for the dosing interval    -   F Fraction available (bioavailability):

F=[AUC_(oral)]·dose_(iv)/[AUC_(iv)]·dose_(oral)

-   -   CL Clearance    -   CLr Renal clearance, calculated for the 24-hour steady-state        period according to

${CLr} = \frac{{Ue}\left( {0 - 24} \right)}{{AUC}\left( {0 - 24} \right)}$

-   -   -   Where Ue is excreted drug

    -   Cl/F Apparent total body clearance as a function of        bioavailability

${{CL}/F} = \frac{Dose}{{AUC}\left( {0 - 24} \right)}$

-   -   V_(ss) Steady state volume of distribution    -   V_(d) Volume of distribution    -   V_(z)/F Apparent terminal phase volume of distribution as a        function of bioavailability

${{Vz}/F} = \frac{Dose}{\lambda \; z \times {{AUC}\left( {0 - 24} \right)}}$

-   -   t_(1/2) Terminal half-life (HL;), calculated by the equation        t½=0.693/k_(el)    -   C_(max) The maximum observed concentration, obtained directly        from the plasma concentration time profile    -   T_(max) The time of C_(max); at more than one time point, the        first is chosen    -   Az elimination rate constant, calculated as the negative of the        slope of the terminal log-linear segment of the plasma        concentration-time curve, where slope is determined from a        linear regression of the natural logarithm of the terminal        plasma concentrations against time; at least 3 terminal plasma        concentration time points, beginning with the final        concentration ≧LOQ, will be selected for the determination of λz        and the regression will need coefficient of determination        (r²)≧0.9000    -   k_(el) The terminal elimination rate constant will be obtained        from the slope of the line, fitted by linear least squares        regression, through the terminal points of the log (base e)        concentration-time profiles.

Typically, actual blood sample collection times relative to the start ofI2S intrathecal administration are used in the IT PK analysis. Forexample, blood samples are typically collected within 15 or 30 minutesprior to I2S intrathecal administration (pre-injection baseline or time0) and at 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, 24, 30, 36, 48, 60, 72, 84,96, 108, 120, 132, 144, 156, 168 or 180 hours following intrathecaladministration. If IT is administered in conjunction with IVadministration, for IV PK analysis, blood samples are collected 15 or 30minutes prior to IV infusion (pre-infusion baseline or time 0) and at0.5, 1, 1.5, 2, 2.5, and 3 hours during the infusion (if the infusion is3 hours long), and at 3.5, 4, 5, 6, 7, 9, 11, and 24 hours following theinitiation of IV infusion.

Various methods may be used to measure I2S protein concentration inserum. As a non-limiting example, enzyme-linked immunosorbant assay(ELISA) methods are used.

Pharmacokinetic parameters for I2S can be determined usingcompartmental, noncompartmental, or population-based (i.e., POP-PR)analysis methods known in the art. In some embodiments, pharmacokineticparameters for I2S are determined by noncompartmental analysis usingPhoenix Version 6.1 (Pharsight Corporation. Mountain View, Calif.).

Pharmacokinetic parameters may be evaluated at any stage during thetreatment, for example, at week 1, week 2, week 3, week 4, week 5, week6, week 7, week 8, week 9, week 10, week 11, week 12, week 13, week 14,week 15, week 16, week 17, week 18, week 19, week 20, week 21, week 22,week 23, week 24, or later. In some embodiments, pharmacokineticparameters may be evaluated at month 1, month 2, month 3, month 4, month5, mouth 6, month 7, month 8, month 9, month 10, month 11, month 12,month 13, month 14, month 15, month 16, month 17, month 18, month 19,month 20, month 21, month 22, month 23, month 24, or later during thetreatment.

Typically, as described in the Examples section, following intrathecaladministration, serum concentrations of I2S increased slowly.

In some embodiments, the systemic bioavailability of I2S followingintrathecal administration ranges from about 20-90% (e.g., about 20-80%,20-75%, 20-70%, 20-65%, 60-60%, 20-55%, 20-50%, 30-90%, 30-80%, 30-75%,30-70%, 30-65%, 30-60%, 30-55%, 30-50%, 40-90%, 40-80%, 40-75%, 40-70%,40-65%, 40-60%, 40-55%, 40-50%, 50-90%, 50-80%, 50-75%, 50-70%, 50-65%,50-60%). In some embodiments, the systemic bioavailability of I2Sfollowing intrathecal administration is or greater than about 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%.

In some embodiments, various dosing regimens described herein (i.e., atherapeutically effective dose, administered regularly at theadministration interval) results in serum AUC_(ss) of the recombinantI2S enzyme within a range from approximately 200,000 min·ng/mL toapproximately 1,000.000 min·ng/mL (e.g., from approximately 250,000min·ng/mL to approximately 900,000 min·ng/mL, from approximately 300,000min·ng/mL to approximately 800,000 min·ng/mL, from approximately 350,000min·ng/mL to approximately 700,000 min·ng/mL, from approximately 400,000min·ng/mL to approximately 600,000 min·ng/mL).

In some embodiments, various dosing regimens described herein (i.e., atherapeutically effective dose, administered regularly at theadministration interval) results in maximum serum concentration(C_(max)) of the recombinant I2S enzyme within a range fromapproximately 60 to approximately 300 ng/mL (e.g., from approximately 70to approximately 250 ng/mL, from approximately 70 to approximately 200ng/mL, from approximately 70 to approximately 150 ng/mL, fromapproximately 80 to approximately 250 ng/mL, from approximately 80 toapproximately 200 ng/mL, from approximately 80 to approximately 150ng/mL, from approximately 90 to approximately 250 ng/mL, fromapproximately 90 to approximately 200 ng/mL, from approximately 90 toapproximately 150 ng/mL).

Reducing GAG Levels

As described above, Hunter syndrome, or Mucopolysaccharidosis 11 (MPSTI), is an X-linked heritable metabolic disorder resulting from adeficiency of the enzyme iduronate-2-sulfatase (I2S). I2S is localizedto lysosomes and plays an important role in the catabolism ofglycosaminoglycans (GAGs) heparan- and dermatan-sulfate. In the absenceof enzyme, these substrates accumulate within cells, ultimately causingengorgement, followed by cellular death and tissue destruction. Due tothe widespread expression of enzyme, multiple cell types and organsystems are affected in MPS II patients

Thus, Hunter syndrome is characterized by an accumulation ofglycosaminoglycans (GAG) in the lysosomes of affected cells includingboth somatic and CNS cells. A patient suffering from or susceptible toHunter syndrome has abnormally high levels of GAG in the CSF, urineand/or blood. For example, in urine, the normal reference range of uGAGlevels, depending on the age, ranges between 57 and 487 ug/mgcreatinine. However, Hunter syndrome patients without treatment may havehigh uGAG levels, e.g., higher than about 1000 μg/mg creatinine, 1050μg/mg creatinine, 1100 μg/mg creatinine, 1150 μg/mg creatinine, 1200μg/mg creatinine, 1250 μg/mg creatinine, 1300 μg/mg creatinine, 1350μg/mg creatinine, 1400 μg/mg creatinine, 1450 μg/mg creatinine, or 1500μg/mg creatinine.

Patients with Hunter syndrome and cognitive impairment, typically alsohave abnormally high levels of GAGs in the CSF. For example, the CSF GAGlevel in healthy children is typically below the lower limit ofquantification (LLOQ) and in young healthy adults, the CSF GAG level istypically between lower than LLOQ to about 95 ng/ml. However, in aHunter syndrome patient, the baseline pre-treatment measurement of theCSF GAG level may be greater than about 300, 400, 500, 600, 700, 800,900, 1000, 1500, or 2000 ng/ml.

Thus, changes from baseline in the concentrations of GAG in the urine,blood and/or CSF may be used as a biomarker indicative of thepharmacodynamics activity and/or efficacy of I2S in vivo. In particular,changes from baseline in the concentrations of GAG in CSF may be used asa biomarker indicative of the pharmacodynamics activity of I2S in CSFafter intrathecal administration or as endpoints for efficacy. Forexample, according to the present invention, a recombinant I2S enzyme isadministered intrathecally at a therapeutically effective dose and anadministration interval for a treatment period sufficient to decreaseglycosaminoglycan (GAG) level in the cerebrospinal fluid (CSF) and/orurine relative to a control. As used herein, the term “decrease,” orequivalent such as “reduce,” or grammatical equivalents, indicate ameasurement of GAG level that is relative to a baseline measurement,such as a measurement in the same individual prior to initiation of thetreatment, or a measurement in a control individual (or multiple controlindividuals) in the absence of the treatment. A “control individual” isan individual afflicted with Hunter syndrome as the individual beingtreated, who is about the same age as the individual being treated (toensure that the stages of the disease in the treated individual and thecontrol individual(s) are comparable).

In some embodiments, intrathecal administration of a recombinant I2Senzyme according to the present invention results in a reduction of theGAG level in CSF, urine and/or blood by about 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,100% or more as compared to a control (e.g., baseline measurement) insome embodiments, intrathecal administration of a recombinant I2S enzymeaccording to the present invention results in a reduction of the GAGlevel in CSF, urine and/or blood by at least 1-fold, 2-fold, 3-fold,4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or 10-fold as compared toa control (e.g., baseline measurement).

In some embodiments, intrathecal administration of a recombinant I2Senzyme according to the present invention results in the GAG level inthe CSF lower than about 1000 ng/ml (e.g., lower than about 900 ng/ml,800 ng/ml, 700 ng/ml, 600 ng/ml, 500 ng/ml, 400 ng/ml, 300 ng/ml, 200g/ml, 100 ng/m, 100 ng/ml, 50 ng/ml, 10 ng/ml, or 1 ng/ml).

In some embodiments, intrathecal administration of a recombinant I2Senzyme according to the present invention results in the GAG level inurine lower than about 1000 μg/mg creatinine (e.g., lower than about 900μg/mg creatinine, 800 μg/mg creatinine, 700 μg/mg creatinine, 600 μg/mgcreatinine, or 500 μg/mg creatinine).

Various methods for measuring the GAG level in CSF or urine are known inthe art and can be used to practice the present invention. Exemplarymethods include, but are not limited to, electro-spray ionization-tandemmass spectrometry (with and without liquid chromatography), HPLC orLC-MS based assays as described in Lawrence R. et al. Nat. Chem. Biol.;8(2):197-204. In some embodiments, the GAG level is measured at the endof each dosing cycle (e.g., at the end of each month following themonthly intrathecal administration), i.e., immediately before the nextdosing. The GAG level may also be measured at the beginning or in themiddle of each dosing cycle (e.g., at the beginning or middle of eachmonth following the monthly intrathecal administration).

In some embodiments, a reduction of the GAG level in CSF describedherein is achieved after a treatment period of at least 1, 2, 3, 4, 5,6, 8, 10, 12, 18, 24, or more months. In some embodiments, a reductionof the GAG level in CSF described herein is achieved after a treatmentperiod of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 years or longer.

In various embodiments, intrathecal administration of a recombinant I2Senzyme may be used to maintain the GAG level in CSF at a low level(e.g., lower than about 1000 ng/ml, 900 ng/ml, 800 ng/ml, 700 ng/ml, 600ug/ml, 500 ng/ml, 400 ng/ml, 300 ng/ml, 200 ng/ml, 100 ng/ml, 50 ng/ml,10 ng/ml, or 1 ng/ml) for more than 3, 4, 5, 6, 8, 10, 12 months, or 1,2, 3, 4, 5, 6, 7, 8, 9, 10, years, or the life-time of the patient beingtreated. In various embodiments, intrathecal administration of arecombinant I2S enzyme may be used to maintain the GAG level in urine ata low level (e.g., lower than about 1000 μg/mg creatinine, 900 μg/mgcreatinine, 800 μg/mg creatinine, 700 μg/mg creatinine, 600 μg/mgcreatinine, or 500 μg/mg creatinine) for more than 3, 4, 5, 6, 8, 10, 12months, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, years, or the life-time of thepatient being treated.

In various embodiments, the GAG level in CSF, urine and/or blood mayalso be used as a biomarker to monitor and/or optimize the treatment.For example, the dose and/or administration interval for intrathecaland/or intravenous administration (if the intrathecal administration isused in conjunction with intravenous administration) may be adjustedbased on the GAG level in the CSF, urine and/or blood. In someembodiments, the dose for intrathecal administration may be increased ifthe GAG level in the CSF or urine or blood fails to decrease relative tothe baseline control after 6, 5, 4, or 3 doses. In particularembodiments, the dose for intrathecal administration may be increased ifthe GAG level in the CSF, urine or blood fails to decrease relative tothe baseline control after 4 doses.

The terms, “improve,” “increase” or “reduce,” as used herein, indicatevalues that are relative to a control. In some embodiments, a suitablecontrol is a baseline measurement, such as a measurement in the sameindividual prior to initiation of the treatment described herein, or ameasurement in a control individual (or multiple control individuals) inthe absence of the treatment described herein. A “control individual” isan individual afflicted with the same disease, who is about the same ageand/or gender as the individual being treated (to ensure that the stagesof the disease in the treated individual and the control individual(s)are comparable).

The individual (also referred to as “patient” or “subject”) beingtreated is an individual (fetus, infant, child, adolescent, or adulthuman) having the disease or having the potential to develop thedisease. In some embodiments, a subject being treated is at least 6moths old, 12 months old, 18 months old, 2 years old, 2.5 years old, 3years old, 3.5 years old, 4 years old, 4.5 years old, or 5 years old. Insome embodiments, a subject being treated is younger than 5, 4.5, 4,3.5, 3, 2.5, 2, or 1.5 years old. In some embodiments, a subject in needof treatment has a GAG level in the CSF greater than about 300, 400,500, 600, 700, 800, 900, 1000, 1500, or 2000 ng/ml before the treatment.In some embodiments, a subject in need of treatment has a GAG level inurine higher than about 1000 μg/mg creatinine, 1050 μg/mg creatinine,1100 μg/mg creatinine, 1150 μg/mg creatinine, 1200 μg/mg creatinine,1250 μg/mg creatinine, 1300 μg/mg creatinine, 1350 μg/mg creatinine,1400 μg/mg creatinine, 1450 μg/mg creatinine, or 1500 μg/mg creatinine.

Other biomarkers of Hunter syndrome may also be used to practice thepresent invention, for example, heparin cofactor II-thrombin complex asdescribed in D. R. Randall et al., “Heparin cofactor II-thrombincomplex: A biomarker of MPS disease,” Molecular Genetics and Metabolism94 (2008) 456-461, the contents of which are hereby incorporated byreference.

Treatment of Cognitive Impairment

A defining clinical feature of Hunter syndrome is central nervous system(CNS) degeneration, which results in cognitive impairment (e.g.,decrease in IQ). Additionally, MRI scans of affected individuals haverevealed white matter lesions, dilated perivascular spaces in the brainparenchyma, ganglia, corpus callosum, and brainstem; atrophy; andventriculomegaly (Wang et al. Molecular Genetics and Metabolism, 2009).The disease typically manifests itself in the first years of life withorganomegaly and skeletal abnormalities. Some affected individualsexperience a progressive loss of cognitive function, with most affectedindividuals dying of disease-associated complications in their first orsecond decade.

Among other things, the present invention may be used to effectivelytreat cognitive impairment in Hunter syndrome patients. In someembodiments, treatment according to the present invention results inimproved cognitive performance of a patient suffering from HuntersSyndrome. As used herein, cognitive performance includes, but is notlimited to, cognitive, adaptive, motor, and/or executive functions.Thus, in some embodiments, a method according to the invention may beused to improve, stabilize or reduce declining of one or more cognitive,adaptive, motor, and/or executive functions relative to a control.

Assessment of Cognitive Performance

Typically, cognitive performance may be assessed by a cognitiveperformance instrument. As used herein, the term “cognitive performanceinstrument” includes a cognitive performance test that can be used toevaluate, classify and/or quantify one or more cognitive, adaptive motorand/or executive functions in a subject. As will be understood by thoseskilled in the art, such a test may be questionnaire or survey filledout by a patient, caregiver, parent, teacher, therapist or psychologist.Exemplary cognitive performance instruments suitable for assessingcognitive, adaptive motor and/or executive functions are describedbelow.

Differential Abilities Scale (DAS-II)

In some specific embodiments, the cognitive performance instrument isthe Differential Ability Scale. The Differential Ability Scale, as thename implies, was developed specifically to be suitable for patientswith various types of impairment. The DAS-II is a cognitive test that isdesigned primarily as a profile test which yields scores for a widerange of abilities, measured either by subtests or composites. However,it has been used as a general test of cognitive ability, including inseverely affected populations. The DAS-II composes 2 overlappingbatteries. The Early Years battery is designed for children ages 2 years6 months through 6 years 11 months. The School-Age Battery is designedfor children ages 7 years 0 months through 17 years 11 months. A keyfeature of these batteries is that they were fully co-normed for ages 5years 0 months through 8 years 11 months. In consequence, children ages7 years 0 months through 8 years 11 months can be given the Early Yearsbattery if that is considered more developmentally appropriate for anindividual than the School-Age Battery. Similarly, more able childrenages 5 years 0 months through 6 years 11 months can be given theSchool-Age Battery. As a result, the test accommodates all 5 to 8 yearold children (i.e., 5 years 0 months through 8 years 11 months) at theextremes of the ability range.

The DAS-II has been validated and normed in the US population and in theBritish population (as the BAS, or British Abilities Scales) A Spanishversion, intended for use in Spain and Spanish-speaking Latin America,is expected to become available in the fall of 2012. The DAS-IIincorporates “tailored testing” to enable examiners to select the mostappropriate items for a child. This has two major advantages First, itenables the measure to be both accurate and very time-efficient, whichis a major advantage for the examiner. Second, it makes testing shorterand less tiring for the child and often enables the child to discontinuea subtest before having experienced a string of consecutive failures—anadvantage for the child, as the tests are more enjoyable and motivating.Without being a limiting example, Table 4 discloses a plurality ofsubtest capable of measuring different cognitive abilities, for asubject undergoing enzyme replacement therapy. FIG. 19 shows the samesubtests and the age ranges at which they are normed.

TABLE 4 List of Cognitive Perfomance Instruments Subtest AbbreviationAbilities Measured Copying Copy Visual-perceptual matching andfine-motor coordination in copying line drawings Early number ENCKnowledge of pre-numerical and numerical concepts concepts Matchingletter-like MLLF Visual discrimination among similar shapes formsMatrices Mat Nonverbal reasoning: perception and application ofrelationships among abstract figures Naming vocabulary NVoc Expressivelanguage; knowledge of names Pattern construction PCon Visual-perceptualmatching, especially of spatial orientation, in copying block patterns.Nonverbal reasoning and spatial visualization in reproducing designswith colored blocks Pattern Construction PCon(A) The same abilities forPattern construction without a time (alt) constraint Phonological PhPKnowledge of sound structure of the English language and the processingability to manipulate sound Picture similarities PSim Nonverbalreasoning shown by matching pictures that have a common element orconcept Rapid naming RNam Automaticity of integration of visual symbolswith phonologically referenced naming Recall of designs RDes Short-termrecall of visual and spatial relationships through reproduction ofabstract figures Recall of digits DigF Short-term auditory memory andoral recall of sequences of forward numbers Recall of digits DigBShort-term auditory memory and oral recall of sequences of backwardnumbers Recall of objects - RObI Short-term recall of verbal andpictorial information Immediate Recall of objects - RObDIntermediate-term recall of verbal and pictorial information DelayedRecall of sequential SeqO Short-term recall of verbal and pictorialinformation order Recognition of RPic Short-term, nonverbal visualmemory measure through recognition pictures of familiar objectsSequential and SQR Detection of sequential patterns in figures ornumbers quantitative reasoning Speed of information SIP Quickness inperforming simple mental operations processing Verbal VCom Receptivelanguage: understanding of oral instructions involving comprehensionbasic language concepts Verbal similarities VSim Verbal reasoning andverbal knowledge Word definitions WDef Knowledge of word meanings asdemonstrated through spoken language

Scales of Independent Behavior-Revised (SIB-R)

In some specific embodiments, the cognitive performance instrument isthe scales of independent behavior-revised. The Scales of independentBehavior-Revised (SIB-R) is a measure of adaptive behavior comprising 14subscales organized into 4 adaptive behavior clusters: (1) Motor skills,(2) Social Interaction/Communication, (3) Personal Living skills and (4)Community and Living skills. For each item, the rater is presented withstatements that ask them to evaluate the ability and frequency withwhich the individual being rated can or does perform, in its entirety, aparticular task without help or supervision. The individual'sperformance is rated on a 4-point Likert scale, with responses including(0): Never or Rarely—even if asked; (i) Does, but not Well—or about onequarter of the time—may need to be asked; (2) does fairly well—or aboutthree quarters of the time—may need to be asked; (3) does verywell—always or almost always without being asked.

It also measures 8 areas of problem behavior. The SIB-R provides normsfrom infancy through to the age of 80 and above. It has been used inchildren with autism and intellectual disability. Some experts considerthat one of the strengths of the SIB-R is that has application for basicadaptive skills and problem behaviors of children with significantcognitive or autistic spectrum disorders and can map to AmericanAssociation of Mental Retardation levels of support. The SIB-R isconsidered to be much less vulnerable to exaggeration than some othermeasures of adaptive behaviors.

Bayley Scales Infant Development

In some embodiments, the evaluation of developmental function may beperformed using one or more developmental performance instruments. Insome embodiments, the developmental performance instrument is the BayleyScales of Infant Development (BSID-III). The Bayley Scales of InfantDevelopment is a standard series of measurements used primarily toassess the motor (fine and gross), language (receptive and expressive),and cognitive development of infants and toddlers, ages 0-3. Thismeasure consists of a series of developmental play tasks and takesbetween 45-60 minutes to administer Raw scores of successfully completeditems are converted to scale scores and to composite scores. Thesescores are used to determine the child's performance compared with normstaken from typically developing children of their age (in months). Theassessment is often used in conjunction with the Social-EmotionalAdaptive Behavior Questionnaire. Completed by the parent or caregiver,this questionnaire establishes the range of adaptive behaviors that thechild can currently achieve and enables comparison with age norms.

Wechsler Intelligence Scale for Children (WISC)

In some embodiments, the Wechsler Intelligence Scale for Children (WISC)may be performed. Typically, the WISC test is an individuallyadministered intelligence test for children, in particular, childrenbetween the ages of 6 and 16 inclusive. In some embodiments, the WISCtest can be completed without reading or writing. An WISC scoregenerally represents a child's general cognitive ability.

Vineland Adaptive Behavior Scales

In some embodiments, Vineland Adaptive Behavior Scales are performed.Typically, Vineland Adaptive Behavior Scales measure a person's adaptivelevel of functioning. Typically, the content and scales of VinelandAdaptive Behavior Scales are organized within a three domain structure:Communication, Daily Living, and Socialization. This structurecorresponds to the three broad Domains of adaptive functioningrecognized by the American Association of Mental Retardation (AAMR,2002): Conceptual, Practical, and Social. In addition, Vineland AdaptiveBehavior Scales offer a Motor Skills Domain and an optional MaladaptiveBehavior Index to provide more in-depth information

Additional exemplary cognitive performance instruments suitable for thepresent invention are listed in FIGS. 19 and 20.

Brain Structure Volume

In addition to various standardized tests described herein, brainstructure volume may be used to assess brain health and function. Forexample, such analysis may be performed by examining total cortical graymatter volume, derived from automated analysis of serial brain magneticresonance imaging scans (MRIs).

Cognitive Improvement

In various embodiments, the present invention provides methods fortreating Hunters syndrome, in particular, by improving cognitiveperformance. For example, a method according to the invention mayinclude a step of administering intrathecally to a subject in need oftreatment a recombinant iduronate-2-sulfatase (I2S) enzyme at atherapeutically effective dose and an administration interval for aperiod sufficient to improve, stabilize or reduce declining of one ormore cognitive, adaptive, motor, and/or executive functions relative toa control. As used herein, the terms “improve,” “stabilize” or “reduce,”or grammatical equivalents, indicate an assessment or measurement ofcognitive, adaptive, motor, and/or executive functions (e.g., cognitivetest scores) that are relative to a baseline assessment or measurement,such as an assessment or measurement in the same individual prior toinitiation of the treatment described herein, or an assessment ormeasurement in a control individual (or multiple control individuals) inthe absence of the treatment described herein. A “control individual” isan individual afflicted with Hunter Syndrome as the individual beingtreated, who is about the same age as the individual being treated (toensure that the stages of the disease in the treated individual and thecontrol individual(s) are comparable).

Various cognitive instruments including those described herein may beused to assess one or more cognitive, adaptive, motor, and/or executivefunctions. In some embodiments, the Differential Ability Scales-SecondEdition (DAS-II) is used. The DAS-II assessment may be presented as araw score, cluster score, standardized score, percentile age equivalent,or developmental quotient. In some embodiments, the DAS-II assessment ispresented as a general conceptual ability (GCA) score. In someembodiments, Bayley Scales of Infant Development Version III (BSID-III)is used.

In various embodiments, intrathecal administration of the recombinantI2S enzyme results in improved GCA score or BSID-III developmentalquotient relative to a control (e.g., baseline pre-treatment score)after a treatment period of or longer than 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 18 months, or 1, 2, 3, 4, 5, 10 years. For example, intrathecaladministration of the recombinant I2S enzyme may improve the GCA scoreor BSID-III developmental quotient by 5, 10, 11, 12, 13, 14, 15, 20, 25,30 points or more as compared to a control (e.g., baseline pr-treatmentscore) after a treatment period of or longer than 3, 4, 5, 6, 7, 8, 9,10, 11, 12, or 18 months. In some embodiments, intrathecaladministration of the recombinant I2S enzyme may improve the (GCA scoreor BSID-III developmental quotient by 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50% or more as compared to a control (e.g., baseline pre-treatmentscore) after a treatment period of or longer than 3, 4, 5, 6, 7, 8, 9,10, 11, 12, or 18 months. In some embodiments, intrathecaladministration of the recombinant I2S enzyme may result in an improvedGCA score or BSID-III developmental quotient within the range of 70-105(e.g., 70-100, 70-95, 70-90, 75-105, 75-100, 75-95, 75-90, 80-105,80-100, 80-95, 80-90) after a treatment period of or longer than 3, 4,5, 6, 7, 8, 9, 10, 11, 12, or 18 months. In some embodiments,intrathecal administrator of the recombinant I2S enzyme may result in animproved GCA score or BSID-III developmental quotient of or greater than70, 75, 80, 85, 86, 87, 88, 89, 90 points, or greater after a treatmentperiod of or longer than 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 18 months.Typically, intrathecal administration of the recombinant I2S enzyme mayalso maintain the improved score for a period of or longer than 3, 6, 9,12, 15, 18, 21, 24, 27, 30, 33, or 36 months. As used herein,maintaining the GCA score or BSID-III developmental quotient means thechange of GCA score or BSID-III developmental quotient is less than 10,9, 8, 7, 6, or 5 points within a period of 3, 6, 8, 10, 12 months or thechange of the GCA score or BSID-III developmental quotient over a periodof 3, 6, 8, 10, 12 months within 20% 15%, 10%, 5% of the mean over suchperiod.

In some embodiments, intrathecal administration of the recombinant I2Senzyme results in stabilization of the GCA score or BSID-IIIdevelopmental quotient relative to the baseline pre-treatment assessmentafter a treatment period of or longer than 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 18 months, or 1, 2, 3, 4, 5, 10 years. As used herein, stabilizationof the GCA score or BSID-III developmental quotient means the change ofGCA score or BSID-III developmental quotient from the baseline is lessthan 10, 9, 8, 7, 6, or 5 points within 3, 6, 8, 10, 12 months or thechange of the GCA score or BSID-III developmental quotient over a periodof 3, 6, 8, 10, 12 months within 20%, 15%, 10%, 5% of the mean over suchperiod. In some cases, stabilization of the GCA score or BSID-IIIdevelopmental quotient means the change of GCA score or BSID-IIIdevelopmental quotient from the baseline is less than 20%, 15%, 10%, 5%within 3, 6, 8, 10, 12 months. In some embodiments, the stabilizationhappens after the initial declining of the GCA score or BSID-IIIdevelopmental quotient. For example, stabilization may follow after noless than 40%, 35%, 30%, 25%, 20%, 15%, or 10% declining of the GCAscore or BSID-III developmental quotient from the baseline. In someembodiments, intrathecal administration of the recombinant I2S enzymemay stabilize the GCA score or BSID-II developmental quotient for aperiod of or longer than 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, or 36months. In some embodiments, intrathecal administration of therecombinant I2S enzyme may stabilize the GCA score or BSID-IIIdevelopmental quotient for a period of 3-36 months (e.g., 3-33, 3-30,3-27, 3-24, 3-21, 3-18, 3-15, 3-12, 3-9, 3-6, 6-36, 6-33, 6-30, 6-27,6-24, 6-21, 6-18, 6-15, 6-12, 6-9 months).

In some embodiments, intrathecal administration of the recombinant I2Senzyme results in reduced declining of the GCAscore or BSID-IIIdevelopmental quotient relative to a control (e.g., the baselinepre-treatment score) after a treatment period of or longer than 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 18 months, or 1, 2, 3, 4, 5, 10 years. Forexample, the intrathecal administration of the recombinant I2S enzymemay result in the annual decline of the GCA score or BSID-IIIdevelopmental quotient less than about 20, 19, 18, 17, 16, 15, 14, 13,12, 11, or 10 points. In some embodiments, the intrathecaladministration of the recombinant I2S enzyme may result in the annualdecline of the GCA score or BSID-III developmental quotient less thanabout 40%, 35%, 30%, 25%, 20%, 15%, or 10%.

In some embodiments, intrathecal administration of the recombinant I2Senzyme further results in improvement or stabilization of one or moreadaptive functions assessed by the Scales of IndependentBehavior-Revised (SIB-R). In some embodiments, intrathecaladministration of the recombinant I2S enzyme further results inimprovement or stabilization of one or more executive functions assessedby the Behavior Rating Inventory of Executive Function® (BRIEF®).

In some embodiments, cognitive improvement described herein is achievedafter a treatment period of at least 3, 4, 5, 6, 8, 10, 12, 18, 24, 30,36, or more months. In some embodiments, cognitive improvement describedherein is achieved after a treatment period of at least 1, 2, 3, 4, 5,6, 7, 8, 9, 10 years or longer.

In various embodiments, intrathecal administration of a recombinant I2Senzyme may be used to maintain the cognitive improvement describedherein for more than 3, 4, 5, 6, 8, 10, 12 months, or 1, 2, 3, 4, 5, 6,7, 8, 9, 10, years, or the life-time of the patient being treated.

In various embodiments, one or more cognitive, adaptive, motor, and/orexecutive functions may also be used as biomarkers to monitor and/oroptimize the treatment. For example, the dose and/or administrationinterval for intrathecal and/or intravenous administration (if theintrathecal administration is used in conjunction with intravenousadministration) may be adjusted (e.g., increasing or decreasing) basedon the GCA, BSID-III, SIB-R, and/or BRIEF score. In some embodiments, ifthe GCA, BSID-II, SIB-R, and/or BRIEF score fails to improve after 4, 5,or 6 doses, the dose for intrathecal administration may be increased.

The terms, “improve,” “increase” or “reduce,” as used herein, indicatevalues that are relative to a control. In some embodiments, a suitablecontrol is a baseline assessment or measurement, such as an assessmentor measurement in the same individual prior to initiation of thetreatment described herein, or an assessment or measurement in a controlindividual (or multiple control individuals) in the absence of thetreatment described herein. A “control individual” is an individualafflicted with the same disease, who is about the same age and/or genderas the individual being treated (to ensure that the stages of thedisease in the treated individual and the control individual(s) arecomparable).

The individual (also referred to as “patient” or “subject”) beingtreated is an individual (fetus, infant, child, adolescent, or adulthuman) having the disease or having the potential to develop thedisease. It is contemplated begin intrathecal therapy early in thetrajectory of neurodevelopmental decline may be particularly effective mtreating cognitive impairment. Thus, in some embodiments, a subjectbeing treated is at least 6 moths old, 12 months old, 18 months old, 2years old, 2.5 years old, 3 years old, 3.5 years old, 4 years old, 4.5years old, or 5 years old. In some embodiments, a subject being treatedis younger than 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5 years old, or 12, 10, 6months old. In some embodiments, a subject being treated is younger than12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 month old. In some embodiments,a subject being treated is younger than 29, 28, 27, 26, 25, 24, 23, 22,21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2,1 day(s) old. In some embodiments, the subject being treated has a GCAscore or BS ID-III developmental quotient less than 100, 90, 80, 70, 60,50, 40, 30, 20, 15, 10 or not testable before the treatment. In someembodiments, the subject being treated has a GCA score or BSID-IIIdevelopmental quotient declined from normal baseline less than 40%, 35%,30%, 25%, 20%, 15%, or 10% before the treatment. In some embodiments,the subject being treated has a GCA score or BSID-III developmentalquotient ranging between about 60-100 (e.g., about 60-95, 60-90, 60-85,60-80, 60-75, 60-70, 70-100, 70-95, 70-90, 70-85, 70-80, 80-100, 80-95,80-90) before the treatment

The invention will be more fully understood by reference to thefollowing examples. They should not, however, be construed as limitingthe scope of the invention. All literature citations are incorporated byreference.

EXAMPLES Example 1: Evaluation of I2S Serum and/or CSF ConcentrationLevels in Pediatric Subjects

The experiments presented in this example were designed to evaluatesuitable models for determining predictive I2S concentration in serumand/or CSF and analyze observed vs. predicted I2S concentration inpediatric subjects following IV or IT dosing.

First, experiments were conducted to investigate various compartmentalmodels and their respective ability to fit serum and CSF concentrationdata after IV and IT-L administration of I2S in human subjects. Meanconcentration-time profiles of I2S following IV and IT-L administrationof various doses of I2S were determined in serum and CSF using standardmethods.

Structure modeling was used to construct a 2 compartment model (FIG. 1),as well as parameters demonstrating intercompartental exchange betweenplasma and CSF (FIG. 2). Predictive analysis for both IV and ITadministration was evaluated using both Human and Allometric models toestimate various I2S parameters in various matrices (e.g., serum andCSF) in children (FIGS. 3 and 4). In order to evaluate the Human model,I2S serum concentration following IV or IT administration in humanpatients, were analyzed using an ELISA assay. As indicated by the data,use of a Human model provides an accurate prediction of I2S serumconcentration for IT and IV administration based on data from non-humanprimate, as compared to observed values.

However, given the difference in height, weight and body mass betweenhumans and non-human primate subjects, additional studies were performedto evaluate use of an Allometric model. Serum concentrations levels ofI2S were measured in both pediatric subjects and non-human primates overvarious time-points, following IT-L delivery. A correction factor wascalculated based on the difference in brain to body weight ratio, forhumans and non-human primates. FIG. 5 shows allometrically scaledpopulation pharmacokinetic (PopPK) parameters to pediatrics for IT-I2Safter correction for brain/BW ratio difference between non-humanprimates (NHP) and children. For both serum and CSF, calculations werecarried out to estimate predictive values for the various parameterswithin two pools of subjects: pediatric subjects less than 6 years ofage and juveniles ages 6-17. Calculations were performed by taking theestimated pharmacokinetic value for each parameter in non-human primatesand correcting for differences, using a brain/body weight ratio ofnon-human primate over child (FIG. 5). An average weight of 2.73 kg wasused for non-human primates. Parameters were scaled using the medianbody weight (20.6 kg) of human clinical pediatric subjects (FIG. 5).FIG. 6, demonstrates an exemplary Elementary Dedrick Plot of BW-Scaledserum I2S concentration vs. scaled time in pediatric subjects andmonkeys after IT-L dosing.

I2S serum concentrations obtained form pediatric subjects were analyzedand evaluated, against the predicted serum concentrations determinedusing the model and methods described above. FIG. 7 illustrates observedvs. predicted serum concentration—time profile of I2S in pediatricsubjects following a single IV infusion of I2S at concentration of 0.5mg/kg. The data suggests that predicted profile after IV administrationwas well-predicted by scaled NC model. FIG. 8 illustrates observed vs.predicted serum concentration—time profile of I2S in pediatric subjectsfollowing a single 10 mg IT-L administration. Exemplary Brain/Bodyweight ratio difference correction data were shown in FIG. 9. Thesefindings show that application of Brain/BW ratio correction improvesprediction. Studies were also performed to determine the optimalsampling conditions and time-points for measuring pharmacokineticparameters in serum and CSF following I2S delivery (FIG. 10).

These results demonstrate that the modeling approach described hereinmay allow accurate prediction of pharmacologic measurements in humansubjects based on data obtained from non-human primates. These resultsalso suggest that IT delivery in human subjects, in particular,pediatric subjects can impact concentration of I2S in both serum and CSFand the serum and/or CSF I2S concentrations can be used to monitorand/or optimize treatment and therapeutic efficacy.

Example 2: Evaluating Tolerance of a 100 mg Dosage and ModelingPredictive I2S Concentration in Serum and CSF

Using both the experimental data and pharmacokinetic models obtainedabove, calculations were performed to evaluate if a monthly 100 mg doseof Idursulfase would be safe and/or offer additional efficacy beyond 30mg dose. Using both a Human model or Allometric model, the projectedplasma exposure, including projected C_(max) and AUC values, anticipatedafter monthly 100 mg IT delivery of Idursulfase can be extrapolated(FIGS. 11 and 12). As such, the data suggests that such an approachcould be used to determine the safety and efficacy of monthly 100 mg ITdose (or higher) of Idursulfase. Furthermore, it suggests that given theprojected pharmacokinetic profile, a 100 mg IT dose could be effectivelyand safely administered to a human patient.

Example 3. Pharmacokinetic Analysis of Intrathecally Administered I2S

This serum pharmacokinetic (PK) properties of a idursulfase-IT(recombinant human I2S for intrathecal [IT] administration) wereevaluated in the phase I/II clinical study designed to evaluate thesafety and efficacy of IT delivery of I2S replacement enzyme in humanpatients with Hunter Syndrome.

Human subjects previously diagnosed with Hunters Syndrome were enrolledin the study and were drawn from a range of age groups with varyingdegrees of disease severity. A purified form of the lysosomal enzymeiduronate-2-sulfatase produced by recombinant DNA technology in a humancell line, was used in the clinical trials. For the study, anintrathecal drug delivery device (IDDD) was implanted in the intrathecalspace surrounding the spinal chord for each subject. Depending on thestudy group, a monthly does of either 0, 1, 10 or 30 mg of idursulfasewas delivered intrathecally through IDDD in combination with IVadministration. The monthly dosing was continued up to a period of 36months to determine drug tolerance and efficacy. The formulation forintrathecal administration used in this study contains I2S (50 mg/ml),sodium chloride (9 mg/ml), and polysorbate 20 (0.00005 ml/ml).

To determine the pharmacokinetic profile of idursulfase in serum samplescollected from pediatric patients two research arms were established.The objective of this two arm approach, was to determine the PK profileof idursulfase m serum samples collected from pediatric patients withHunter syndrome and with cognitive impairment who receivedidursulfase-IT by the intrathecal route at monthly intervals inconjunction with IV administration of idursulfase (3 hr 0.5 mg/kginfusion) at weekly intervals. The initial research arm was arandomized, multicenter, multiple-dose, time-lagged, dose escalationstudy evaluating the safety, tolerability, and clinical activity of upto 4 dose levels of idursulfase-IT administered via an intrathecal drugdelivery device (IDDD) monthly for 6-months in conjunction with weeklyintravenous (IV) infusions of recombinant I2S (0.5 mg/kg) in patientswith Hunter syndrome and who have cognitive impairment. Patients whocompleted all study requirements for the first arm were then allowed toparticipate in a second Interim Study, which was designed as anopen-label extension of the initial study, to evaluate the long-termsafety and clinical outcomes of idursulfase-IT administered inconjunction with intravenous I2S administration in pediatric patientswith Hunter Syndrome and cognitive impairment.

There were four patients per treatment group enrolled in the initialresearch arm. An additional group of 4 patients were randomly assignedto no-IT treatment for 6-months (an IV-only-group within the IT and IVtreatment groups). Patients were enrolled in Group 1 (10 mg) and Group 2(30 mg) in a sequential, escalating fashion. Enrollment in Group 4 (1.0ng) commenced following Group 2 enrollment. Due to the favorable PDeffect observed at 30 mg, the initially planned 100 mg dose was notimplemented and a 1 mg dose group was implemented instead.

The duration of idursulfase-IT treatment in the initial arm was 6months, with patients receiving 1 dose of idursulfase-IT every 28 days.Patients who completed all study requirements in the initial arm went onto participate in an open-label extension study to evaluate thelong-term safety and clinical outcomes of IT administration ofidursulfase-IT. Patients who received idursulfase-IT in the first armreceived the same treatment regimen in the extension study and willcontinue to receive treatment for a maximum duration of 5 years.

For the initial research arm, serum pharmacokinetic analyses wereperformed at week 3 (following the first idursulfase-IT administration)and Week 23 (following the sixth idursulfase-IT administration). Forthose patients who continued on to the second arm of the study,pharmacokinetic analysis was also performed, but at month 19 and month31. During the IT administration weeks, in both the first and secondresearch arms, the IV dose is administered 2 days following the IT dose.

Blood Sampling

Evaluation of idursulfase concentration-time profiles from thecerebrospinal fluid (CSF) is difficult due to the limited number ofclinically permissible CSF sample collections. Therefore, whiledetermination of a comprehensive pharmacokinetic profile in CSF was notpossible, idursulfase levels were evaluated indirectly by measuringsystemic serum idursulfase concentration-time profiles, as sampled fromthe blood. Blood samples were collected from patients who receivedidursulfase-IT or IV at Weeks 3 and 23 (10 and 30 mg group) and Month 19(10 mg group) and analyzed. All blood samples for PK analysis were drawnfrom a vessel in the arm opposite from IV infusion and placed incollection tubes without any anticoagulant and were allowed to clot atroom temperature. Blood samples were collected within 15 minutes priorto idursulfase-IT administration (pre-injection baseline or Time 0) andat 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, and 24 hours following idursulfase-ITadministration. Sampling times were extended to 30 and 36 hoursfollowing idursulfase-IT administration at Week 23 (30 mg group) and atMonth 19. Blood samples for IV PK evaluation were collected within 15minutes prior to IV infusion (pre-infusion baseline or Time 0), at 0.5,1, 1.5, 2, 2.5, and 3 hours during the infusion; and at 3.5, 4, 5, 6, 7,9, 11, and 24 hours following the initiation of IV infusion.

Analysis of Serum I2S

Serum samples for PK analysis were analyzed for idursulfase proteinconcentration using validated enzyme-linked immunosorbant assay (ELISA)methods. The lower limit of quantification (LLOQ) of the ELISA methodused to measure serum idursulfase concentrations after IV administrationwas 62.5 ng/mL. The LLOQ of the assay used to measure serum idursulfaseafter idursulfase-IT administration was 6.25 ng/mL. A higher sensitivityidursulfase protein assay was used for the idursulfase-IT samples inorder to detect and examine anticipated lower amounts of idursulfaseentering systemic circulation from the CNS compartment following ITadministration.

Pharmacokinetic Analysis

Pharmacokinetic parameters for idursulfase were determined bynoncompartmental analysis using Phoenix Version 6.1 (PharsightCorporation, Mountain View, Calif., USA) The actual PK blood samplecollection times relative to the start of idursulfase-IT administrationwere used in the PK analysis. For the PK analysis of serum idursulfaseconcentrations after the start of IV infusion, the actual infusion times(approximately 180 min) and actual sampling times were used. Continuousdata were summarized with the descriptive statistics: number ofobservations, mean, standard deviation (SD), geometric mean, coefficientof variation (% CV), median, minimum, and maximum values. Categoricaldata were summarized with frequencies and/or percentages. Thepharmacokinetic parameters calculated for each sample included: maximumobserved serum concentration (C_(max)), time of C_(max) (T_(max)), areaunder the serum concentration-time curve from time zero to the lastsampling time at which serum concentrations were measurable(AUC_(0-last)), area under the serum concentration-time curveextrapolated to infinity (AUC_(0-∞)), exposure at steady state for thedosing interval (AUC_(ss)), terminal rate constant (λz) derived from theslope of the log-linear regression of the log-linear terminal portion ofthe serum concentration-time curve, terminal half-life (t½) calculatedas 0.693/λz, mean residence time extrapolated to infinity (MRT_(inf));which is calculated as AUMC_(0-∞)/AUC_(0-∞), total clearance (CL)calculated as dose/AUC_(0-∞), volume of distribution (V_(ss)) calculatedas MRT_(inf)-CL and distribution of volume (V_(z)) derived from theelimination phase.

I2S-IT and IV (Week 3)

The data demonstrates that at doses of 10 and 30 mg, idursulfase-ITexhibited similar serum idursulfase concentration-time profiles (FIG.13). Intrathecal administration of idursulfase-IT demonstrated a slowdistribution into the systemic compartment, with a maximum observedconcentration (T_(max)) for the 10 mg and 30 mg idursulfase-IT doses of545.5±226.1 minutes and 420±84.9 minutes, respectively. At Week 3 therewas a high degree of variability in the C_(max) and AUC_(0-last) valuesof individual patients in the 10 mg (n=4) and 30 mg (n=2) idursulfase-ITgroups, but in general, systemic exposure was similar for the twotreatment groups. The C_(max) was 144.5±65.9 g/mL and 204.8±33. ng/mL,and the AUC_(0-last) was 140084.5±45590.1 min·ng/mL and 190487.7±38569.0min·ng/mL for the 10 and 30 mg idursulfase-IT groups, respectively(Table 5). The IV AUC_(0-∞) and C_(max) for the idursulfase-IT 10 mggroup at Week 3 was 469936.2±85471.3 min·ng/mL and 1695.5±376.0 ng/mL,respectively. The I2S IV AUC_(0-∞) and C_(max) for the idursulfase-IT 30mg group at Week 3 was 553300.4±190671.0 min·ng/mL and 2187.5±979.5ng/mL, respectively (Table 8).

I2S-IT and IV (Week 23)

At Week 23 the PK profiles of both idursulfase-IT dose groups weresimilar to Week 3 (FIG. 14). Idursulfase-IT exhibited slow distributioninto the systemic compartment, with a T_(max) of 570.8±181.5 minutes and450.5±60.3 minutes, for the 10 mg and 30 mg doses respectively. Similarto Week 3, at Week 23 there was a high degree of variability in C_(max)and AUC_(0-last) values of individual patients in the 10 mg (n=4) and 30mg (n=4) idursulfase-IT groups and systemic exposure was higher for the10 mg idursulfase-IT group. The C_(max) was 150.4±50.2 ng/mL and95.1±59.3 ng/mL, and AUC_(0-last) was 150529.0±43878.8 min·ng/mL and102278.3±105526.2 min·ng/mL for the 10 mg and 30 mg idursulfase-ITgroups, respectively (Table 6). At Week 23, the I2S IV AUC_(0-∞) andC_(max) for the idursulfase-IT 10 mg group was 483492.6±69182.3min·ng/mL and 1704.7±410.0 ng/mL, respectively. The I2S IV AUC_(0-∞) andC_(max) for the idursulfase-IT 30 mg group at Week 23 were546934.2±115402.7 min·ng/mL and 2142.1±660.9 ng/mL, respectively (Table9).

I2S-IT and IV (Month 19)

At the Month 19 timepoint, the PK profile of idursulfase-IT is similarto that observed at Week 3 and 23 (FIG. 15). Serum concentrations ofidursulfase had a T_(max) of 570.0±180.0 minutes. Evaluation at Month 19shows that the systemic exposure at the 10 mg dose of idursulfase-IT wascomparable to the values observed at Week 3 and Week 23. The C_(max) andAUC_(0-last) were 96.4±44.3 ng/mL and 124433.3±30757.6 min·ng/mL,respectively (Table 7). Intravenously administered I2S (0.5 mg/kg)exhibited overlapping serum idursulfase concentration-time profiles(FIG. 3) as well as similar PK parameters at Week 3 and Week 23 (Table9). At both time periods, the C_(max) generally coincided with the endof infusion (3 hours).

TABLE 5 Exemplary Noncompartmental PK Parameters of Serum IdursulfaseConcentrations from Patients in the First Arm Following Administrationof Idursulfase-IT (Week 3) t_(1/2) T_(max) C_(max) AUC_(0-last)AUC_(0-∞) V_(s) CL MRT_(inf) Patient (min) (min) (ng/mL) (min · ng/mL)(min · ng/mL) (mL) (mL/min) (min) 10 mg 045-013-0004 1308.2 250.0 134.1147351.1 295976.6 63765.4 33.8 2018.5 045-013-0005 NC 725.0 91.2100284.5 NC NC NC NC 045-013-0011 NC 720.0 112.9 111243.9 NC NC NC NC045-013-0014 NC 487.0 239.9 201458.4 NC NC NC NC N 1 4 4 4 1 1 1 1 Mean1308.2 545.5 144.5 140084.5 295976.6 63765.4 33.8 2018.5 SD NC 226.165.9 45590.1 NC NC NC NC CV % NC 41.5 45.6 32.5 NC NC NC NC Median1308.2 603.5 123.5 129297.5 295976.6 63765.4 33.8 2018.5 Min 1308.2250.0 91.2 100284.5 295976.6 63765.4 33.8 2018.5 Max 1308.2 725.0 239.9201458.4 295976.6 63765.4 33.8 2018.5 Geo_(mean) 1308.2 502.1 134.9134900.1 295976.6 63765.4 33.8 2018.5 30 mg 045-013-0003 622.4 360.0181.3 163215.3 217883.3 123639.1 137.7 1073.6 045-013-0006 NC 480.0228.4 217760.1 NC NC NC NC N 1 2 2 2 1 1 1 1 Mean 622.4 420.0 204.8190487.7 217883.3 123639.1 137.7 1073.6 SD NC 84.9 33.3 38569.0 NC NC NCNC CV % NC 20.2 16.3 20.2 NC NC NC NC Median 622.4 420.0 204.8 190487.7217883.3 123639.1 137.7 1073.6 Min 622.4 360.0 181.3 163215.3 217883.3123639.1 137.7 1073.6 Max 622.4 480.0 228.4 217760.1 217883.3 123639.1137.7 1073.6 Geo_(mean) 622.4 415.7 203.5 188525.3 217883.3 123639.1137.7 1073.6 NC—not calculated due to insufficient data points

TABLE 6 Exemplary Noncompartmental PK Parameters of Serum IdursulfaseConcentrations from Patients in the First Arm Following Administrationof Idursulfase-IT (Week 23) t_(1/2) T_(max) C_(max) AUC_(0-last)AUC_(0-∞) V_(s) CL MRT_(inf) Patient (min) (min) (ng/mL) (min · ng/mL)(min · ng/mL) (mL) (mL/min) (min) 10 mg 045-013-0004 1461.8 359.0 220.0214132.4 434076.7 485851 23.0 2128.6 045-013-0005 NC 480.0 116.2120864.6 NC NC NC NC 045-013-0011 NC 724.0 154.1 145280.2 NC NC NC NC045-013-0014 NC 720.0 111.4 121839.0 NC NC NC NC N 1 4 4 4 1 1 1 1 Mean1461.8 570.8 150.4 150529.0 434076.7 48585.1 23.0 2128.6 SD NC 181.550.2 43878.8 NC NC NC NC CV % NC 31.8 33.3 29.1 NC NC NC NC Median1461.8 600.0 135.1 133559.6 434076.7 48585.1 23.0 2128.6 Min 1461.8359.0 111.4 120864.6 434076.7 48585.1 23.0 2128.6 Max 1461.8 724.0 220.0214132.4 434076.7 48585.1 23.0 2128.6 Geo_(mean) 1461.8 547.5 144.7146299.7 434076.7 48585.1 23.0 2128.6 30 mg 045-013-0003 656.1 480.0182.0 235827.9 274312.5 103513.1 109.4 1165.9 045-013-0006 3898.6 360.075.0 137590.2 474893.4 355313.4 63.2 5841.3 045-014-1007 NC 482.0 48.412024.5 NC NC NC NC 045-014-1009 NC 480.0 75.1 23670.5 NC NC NC NC N 2 44 4 2 2 2 2 Mean 2277.3 450.5 95.1 102278.3 374602.9 229413.2 86.33503.6 SD 2292.8 60.3 59.3 105526.2 141832.1 178049.7 32.7 3306.0 CV %100.7 13.4 62.3 103.2 37.9 77.6 37.9 94.4 Median 2277.3 480.0 75.180630.3 374602.9 229413.2 86.3 3503.6 Min 656.1 360.0 48.4 12024.5274312.5 103513.1 63.2 1165.9 Max 3898.6 482.0 182.0 235827.9 474893.4355313.4 109.4 5841.3 Geo_(mean) 1599.3 447.2 83.9 55127.0 360928.2191780.1 83.1 2609.7 NC—not calculated due to insufficient data points

TABLE 7 Exemplary Noncompartmental PK Parameters of Serum IdursulfaseConcentration from Patients in the Second Arm Following Administrationof Idursulfase-IT (Month 19) t_(1/2) T_(max) C_(max) AUC_(0-last)AUC_(0-∞) V_(s) CL MRT_(inf) Patient (min) (min) (ng/mL) (min · ng/mL)(min · ng/mL) (mL) (mL/min) (min) 10 mg 046-013-0004 NC 480.0 159.1166524.2 NC NC NC NC 046-013-0005 1413.3 360.0 94.6 119038.8 190710.6106917.3 52.4 2183.7 046-013-0011 1092.2 720.0 58.8 92593.8 135247.7116503.9 73.9 1860.3 046-013-0014 1162.1 720.0 73.1 119576.3 180115.593080.3 55.5 1969.6 N 3 4 4 4 3 3 3 3 Mean 1222.5 570.0 96.4 124433.3168691.3 105500.5 60.6 2004.5 SD 168.9 180.0 44.3 30757.6 29443.511775.9 11.6 164.5 CV % 13.8 31.6 46.0 24.7 17.5 11.2 19.2 8.2 Median1162.1 600.0 83.8 119307.6 180115.5 106917.3 55.5 1969.6 Min 1092.2360.0 58.8 92593.8 135247.7 93080.3 52.4 1860.3 Max 1413.3 720.0 159.1166524.2 190710.6 116503.9 73.9 2183.7 GeoMean 1215.0 547.1 89.7121716.2 166859.9 105054.7 59.9 2000.1 NC—not calculated due toinsufficient data points

TABLE 8 Exemplary Noncompartmental PK Parameters of Serum IdursulfaseConcentrations from Patients in the First Arm Following Administrationof Recombinant I2S IV (Week 3) t_(1/2) T_(max) C_(max) AUC_(0-last)AUC_(0-∞) V_(s) CL MRT_(inf) Vss Patient (min) (min) (ng/mL) (min ·ng/mL) (min · ng/mL) (mL) (mL/min) (min) (mL/kg) Elaprase IV (0.5 mg/kg)in 10 mg idursulfase-IT dosing arm 045-013-0004 769.4 150.0 1211.6292786.7 362161.9 1532.5 1.38 769.0 1061.7 045-013-0005 612.8 150.01642.8 415675.4 470928.8 938.6 1.06 559.2 593.7 045-013-0011 688.2 152.01816.7 413324.4 475376.1 1044.3 1.05 582.6 612.7 045-013-0014 357.5180.0 2110.9 535739.4 571277.9 451.4 0.88 435.6 381.2 N 4 4 4 4 4 4 4 44 Mean 607.0 158.0 1695.5 414381.4 469936.2 991.7 1.1 586.6 662.3 SD178.2 14.7 376.0 99189.8 85471.3 443.4 0.2 137.6 236.1 CV % 29.4 9.322.2 23.9 18.2 44.7 19.3 23.5 43.2 Median 650.5 151.0 1729.8 414499.9473152.4 991.4 1.1 570.9 603.2 Min 357.5 150.0 1211.6 292736.7 362161.9451.4 0.9 435.6 381.2 Max 769.4 180.0 2110.9 535739.4 571277.9 1532.51.4 769.0 1061.7 Geomean 583.6 157.5 1662.2 405170.2 463912.1 907.5 1.1574.7 619.5 Elaprase IV (0.5 mg/kg) in 30 mg idursulfase-IT dosing arm045-013-0003 719.7 180.0 1739.4 454701.0 519597.0 999.2 0.96 589.0 566.8045-013-0006 637.9 120.0 3310.9 672529.5 758575.8 606.6 0.66 557.4 367.4045-014-1009 511.4 182.0 1512.1 335613.3 381728.3 966.4 1.31 544.2 712.8N 3 3 3 3 3 3 3 3 3 Mean 623.0 160.7 2187.5 487614.6 553300.4 857.4 1.0563.6 549.0 SD 104.9 35.2 979.5 170852.6 190671.0 217.8 0.3 23.0 173.4CV % 16.8 21.9 44.8 35.0 34.5 25.4 33.3 4.1 31.6 Median 637.9 180.01739.4 454701.0 519597.0 966.4 1.0 557.4 566.8 Min 511.4 120.0 1512.1335613.3 381728.3 606.6 0.7 544.2 367.4 Max 719.7 182.0 3310.9 672529.5758575.8 999.2 1.3 589.0 712.8 Geomean 616.9 157.8 2057.4 468193.6531871.4 836.7 0.9 563.2 529.5

TABLE 9 Exemplary Noncompartmental PK Parameters of Serum IdursulfaseConcentrations from Patients in the First Study Following Administrationof Recombinant I2S IV (Week 23) t_(1/2) T_(max) C_(max) AUC_(0-last)AUC_(0-∞) V_(s) CL MRT_(inf) Vss Patient (min) (min) (ng/mL) (min ·ng/mL) (min · ng/mL) (mL) (mL/min) (min) (mL/kg) Elaprase IV (0.5 mg/kg)in 10 mg idursulfase-IT dosing area 045-013-0004 904.3 122.0 1448.3386358.5 487860.2 1337.1 1.02 858.4 879.8 045-013-0005 848.6 150.01324.3 312708.6 389225.1 1572.7 1.28 800.2 1027.9 045-013-0011 576.4150.0 1809.5 449898.1 501870.5 828.5 1.00 539.8 537.7 045-013-0014 605.4181.0 2236.5 500429.9 555014.6 786.8 0.90 525.2 473.1 N 4 4 4 4 4 4 4 44 Mean 733.7 150.8 1704.7 412348.8 483992.6 1131.3 1.1 680.9 729.6 SD166.9 24.1 410.0 81182.7 69182.3 386.3 0.2 173.1 267.2 CV % 22.7 16.024.1 19.7 14.3 34.1 15.6 25.4 36.6 Median 727.0 150.0 1628.9 418128.3494865.3 1082.8 1.0 670.0 708.8 Min 576.4 122.0 1324.3 312708.6 389225.1786.8 0.9 525.2 473.1 Max 904.3 181.0 2236.5 500429.9 555014.6 1572.71.3 858.4 1027.9 Geomean 719.3 149.3 1669.1 406113.0 479565.7 1082.0 1.0664.3 692.6 Elaprase IV (0.5 mg/kg) in 30 mg idursulfase-IT dosing arm045-013-0003 1057.0 152.0 1452.7 330584.3 425895.3 1790.3 1.17 928.91090.5 045-013-0006 619.0 187.0 2770.1 588480.6 655723.4 680.9 0.76531.4 405.2 045-014-1007 86.1 210.0 2203.7 507196.8 559184.0 111.1 0.89226.2 202.3 N 3 3 3 3 3 3 3 3 3 Mean 587.4 183.0 2142.1 475420.6546934.2 860.8 0.9 562.2 566.0 SD 486.2 29.2 660.9 131851.9 115402.7853.9 0.2 352.4 465.5 CV % 82.8 16.0 30.9 27.7 21.1 99.2 22.3 62.7 82.2Median 619.0 187.0 2203.7 507196.8 559184.0 680.9 0.9 531.4 405.2 Min86.1 152.0 1452.7 330584.3 425895.3 111.1 0.8 226.2 202.3 Max 1057.0210.0 2770.1 588480.6 655723.4 1790.3 1.2 928.9 1090.5 Geomean 383.3181.4 2069.8 462094.0 538508.8 513.5 0.9 481.5 447.1

Bioavailability of Idursulfase-IT Following IT Administration

The systemic bioavailability of idursulfase-IT after intrathecaladministration was calculated on a subset of patients who had measurableAUC_(0-∞) values (n=6; Table 10). A high degree of inter-patientvariability was observed across the 10 and 30 mg idursulfase-IT dosegroups. The mean percent bioavailability for the 10 mg and 30 mgidursulfase-IT groups was 53.2 (range of 29.9 to 88.0%) and 38.4 (rangeof 24.4 to 59.2%), respectively. The average bioavailability ofidursulfase-IT across both dose groups was 47.7±20.8%.

Thus, following the 10 and 30 mg IT doses, serum concentrations ofidursulfase increased slowly, indicating there was little or no leakageof the intrathecally injected idursulfase and no direct distributioninto systemic circulation. Without wishing to be bound by a particularstudy, one possible mechanism is that idursulfase is removed from theCSF through the arachnoid villi. Materials transverse the villi bymicro-pinocytosis, which is a unidirectional process mediating transportfrom the CSF to the venous system or the epidural space.

The finds also suggest, that during the first (Weeks 0-23) arm of thestudy, the 10 mg and 30 mg doses of idursulfase-IT at Week 3 and Week 23exhibited nearly overlapping serum idursulfase concentration-timeprofiles. The average systemic bioavailability of idursulfase-ITfollowing IT doses of 10 and 30 mg was approximately 48% (range 24-88%).Dose proportionality of serum idursulfase exposure was not observedbetween these two idursulfase doses, with respect to C_(max) orAUC_(0-last), suggesting that saturation of the transfer mechanism(s)from the CNS to the systemic compartment is achieved at an IT dose lessthan or equal to 10 mg.

Safety Profile

Nine of the 12 treated patients (3 of 4 patients in each IT dose group)reported at least one adverse event that was assessed as related toidursulfase-IT. However, no serious adverse events were consideredrelated to idursulfase-IT. There were no deaths during the study, and nopatient experienced a life-threatening adverse event or discontinued dueto an adverse event. Taken together, the clinical data confirms that ITadministration of recombinant I2S enzyme was safe and well tolerated.

Example 4: Intrathecal Administration of Recombinant I2S Reduces GAGLevels in Cerebrospinal Fluid

MPSII (Hunter Syndrome), in its severe form, is characterized by theincrease in the accumulation of GAG within the tissues of the body.Diagnosis of Hunter syndrome is correlated with the onset of progressivedevelopmental delays, especially in adolescent patients. A Phase I/IIsafety trial of intrathecal enzyme replacement with idursulfase-IT usinga formulation of idursulfase, has recently been completed for patientsdiagnosed with Hunter syndrome and suffering from sever cognitiveimpairment. As described in Example 3, sixteen children with MPSII andcognitive impairment were enrolled in 4 dose groups (no treatment, 1 mg,10 mg, 30 mg). Idursulfase-IT was administered monthly for 6 consecutivemonths as a slow bolus via an intrathecal drug delivery device or vialumbar puncture, in conjunction with weekly intravenous infusion (0.5mg/kg). Idursulfase-IT was generally well tolerated. There were no signsof meningeal inflammation due to contact with idursulfase-IT.

The levels of glycosaminoglycans (GAGs) in the cerebrospinal fluid (CSF)were measure using an enzymatic assay. GAG levels were measured duringscreening, during implant surgery, at every monthly dose administration,and at the end of the study. As demonstrated in FIGS. 16-18, prior tothe start of enzyme replacement therapy, all patients had CSF GAG levelsthat were significantly elevated over the levels seen in healthy youngadult volunteers or pediatric controls. In the untreated patients, thelevels remained stable over a 6 month period. Administration ofidursulfase-IT induced a reduction of CSF GAG levels in all treatedpatients (FIGS. 16-18). Furthermore, the findings suggest that stableI2S levels were typically reached after 2 or 3 injections for the 10 and30 mg idursulfase-IT treatment (FIG. 19) No rebound of CSF GAG levelswas observed when a dose was missed (Data not shown).

These data indicate that idursulfase-IT was pharmacodynamically activewhen administered into the CSF of children with MPSII and caneffectively reduce the GAG levels in CSF.

Example 5: Intrathecal Administration of Recombinant I2S ImprovesCognitive Performance

This example demonstrates that intrathecal administration of recombinantI2S enzyme improves cognitive performance in patients diagnosed withHunter syndrome and suffering from severe cognitive impairment based onthe data from a Phase I/II safety trial of intrathecal enzymereplacement with idursulfase-IT (see Example 3)

Intravenous Enzyme Replacement Therapy with recombinant idursulfase isnot expected to affect the cognitive impairment due to theimpenetrability of the blood-brain barrier to large proteins. For thestudy, 4 patients each received 1, 10 or 30 mg idursulfase-IT monthly,with exposures between 6 to 35 months, and 4 additional childrenreceived no-treatment for 6 months and then were switched to activetherapy. Drug was administered via an intrathecal drug delivery deviceor via lumbar puncture. Of the 16 patients, the majority had advancedneurodegenerative disease at enrollment, rendering detailed cognitiveand functional assessments impossible. No Serious Adverse Events relatedto idursulfase-IT have been observed to date.

General Conceptual Ability (GCA) Assessed by DAS-II

Neurodevelopmental testing of children with MPS II typically showsnormal results for the first 2 to 3 years of life; however, at around 3to 4 years of age, those children who will manifest a developmentaldelay start to deviate from the normal developmental trajectory anddecline rapidly over the course of a few years, generally between theages of 3 to 9 years. The sponsor has collected longitudinal data usingthe DAS-II, in the absence of treatment with idursulfase-IT, in MPS IIpatients with evidence of cognitive impairment. These data werecollected in a non-interventional screening study in MPS II patients,and in the period prior to treatment in this first-in-human study. Thedata suggest an annual decline of 13 to 14 points in the GeneralConceptual Ability (GCA) of patients. The GCA score has an average of100 points and a standard deviation of 15 points in healthy children;therefore, an annual decline of 13 to 14 points represents a seriousdeterioration. These data are aligned with other prospectively collecteddata in the published literature.

In this Phase I/II study, the clinical activity of intrathecallyadministered idursulfase-IT, in conjunction with IV therapy, on patientneurodevelopmental status was assessed over 6 months using standardizedmeasures of cognitive, adaptive, motor, and executive functionappropriate for use in children with Hunter syndrome. After completionof baseline assessments, 7 of 12 patients treated with idursulfase-ITwere not capable of being tested serially using the DAS-II instrument tomeasure their neurocognitive function over time. That these patientslacked sufficient neurocognitive function to complete serial assessmentswas due largely to the study inclusion criteria allowing for enrollmentof severely affected patients with established neurocognitiveimpairment, and was a consequence of the study being designed primarilyfor the evaluation of safety, rather than efficacy. Of the 4 patients inthe no-treatment arm, 3 patients were not testable using the DAS-II atthe end of 6 months, and the assessor was not available at theend-of-study visit for testing of 1 of the untreated patients.

Longitudinal assessments using the Differential Abilities Test 2^(nd)version (DAS-II) were obtained in 5 patients, with follow-up timesvarying from 6 months to 24 months. Four of these patients, who received10 or 30 mg idursulfase-IT, showed a stable or higher General ConceptualAbility standard score of the DAS-IL. In particular, one child with afamily history of severe Hunter syndrome maintained his score for up to2 years after initiation of intrathecal enzyme replacement therapy.

A summary of exemplary results of neurocognitive testing is presented inTable 10. The data include both the General Conceptual Ability (GCA)score, as measured by the DAS-II and the Developmental Quotient (DQ), asmeasured by the BSID-III. The main cognitive test utilized during thestudy was the GAS-II; the BSID-III was a fallback measure for use inmore severely affected children. Each cognitive assessment initiatedwith an attempt to perform the GAS-II; however, if the child failed eventhe simplest questions of the GAS-II, the BSID was used as analternative.

As shown in Table 10, of the 5 patients who could be assessed seriallyby the GAS-II, 3 showed evidence of stabilization of neurocognitiveability after 6 months of treatment with idursulfase-IT at the 10 mg or30 mg doses. A fourth patient (at 10 mg) showed varying results duringthe study, and the fifth patient who had received 1 mg, did experience acognitive decline during the 6 months duration of the study.

TABLE 10 Summary of Neurocognitive Test Results (DAS-II GCA or BSID-IIIDQ) Patient Number Dose Baseline^(a) Week 3^(b) Week 15 Week 27 ExaminerComments 045-013-0004 10 mg MD 74 63 79 DAS-II 045-013-0011 10 mg 47 3341 36 DAS-II Hard to test 045-013-0005 10 mg 46 MD MD MD DAS-II Nottestable 045-013-0014 10 mg 70 69 79 76 DAS-II Stabilized, doing well045-013-0006 30 mg 41 MD MD MD DAS-II Not testable 045-013-0003 30 mg 5963 54 62 DAS-II Stabilization after documented decline 045-014-1009 30mg 43 (DQ) 40 (DQ) MD 44 (DQ) BSID-III 045-014-1007 30 mg 22 (DQ) 13(DQ) 19 (DQ) MD BSID-III 045-013-0017  1 mg 66 50 45 41 DAS-II Severelyaffected 045-014-1008  1 mg 47 (DQ) 49 (DQ) 46(DQ) 43 (DQ) BSID-III045-014-1006  1 mg 16 (DQ) 15 (DQ) MD 12 (DQ) BSID-III 045-013-0024  1mg MD MD MD MD Not testable 045-013-0007 No 49 MD NA MD Not testabletreatment 045-013-0019 No 34 32 NA MD Assessor not available treatmentat the end-of-study visit 045-013-0021 No MD MD NA MD Not testabletreatment 045-014-1001 No 19 MD NA MD Not testable treatment^(a)Baseline is the closest screening measurement before therandomization date. ^(b)For treated patients, the assessment wasperformed after the device had been implanted, but prior to firstidursulfase-IT dose. Abbreviations: DQ = developmental quotient; MD =missing data, either test was not attempted or child could notcooperate; NA = Not Applicable.

Several children in the study could not undergo cognitive assessment atall, or could only be assessed using the BSID-II (Table 10).

Several patients also showed evidence of stabilization or improvement inadaptive (assessed using the SIB-R instrument) and executive function(assessed using the BRIEF) behaviors after receiving 6 months oftreatment with idursulfase-IT.

It is expected that a clearer demonstration of clinical benefit ofintrathecal idursulfase-IT therapy on preservation of neurodevelopmentalfunction may be more evident with longer duration of treatment inpatients who begin IT therapy early in the trajectory ofneurodevelopmental decline.

Broad Independence Assessed by SIB-R

An assessment of Broad Independence (BI) was measured over time usingthe Scale of Independent Behavior-Revised (SIB-R). The BroadIndependence Score is derived like an IQ score, with a populationaverage of 100 and a standard deviation of 15. Exemplary results areshown in Table 11. After treatment, an improvement in Broad IndependenceScores was noted in several patients.

Individual patient plots by chronological age of other subdomains ofadaptive behaviors comprising the SIB-R (e.g., motor skills, socialinteraction/communication skills, personal living skills, communityliving skills) were generally similar to that of broad independenceskills (data not shown).

TABLE 11 Summary of Neurodevelopmental Test Results (SIB-R BI) Patientnumber Dose Baseline^(a) Week 3^(b) Week 15 Week 27 Examiner Comments045-013-0004 10 mg 68 77 MD 93 045-013-0011 10 mg 52 24 24 18045-013-0005 10 mg 38 54 33 30 045-013-0014 10 mg 58 54 70 70045-013-0006 30 mg 29 18 21 13 045-013-0003 30 mg 53 53 50 50045-014-1009 30 mg 17 17 MD 23 045-014-1007 30 mg 14 ND ND  1045-013-0017  1 mg 56 59 MD 44 045-014-1008  1 mg 61 63 52 52045-014-1006  1 mg 11 14 ND 14 045-013-0024  1 mg 40 MD 15 ND045-013-0007 No MD MD NA MD Patient cognitive treatment limitations045-013-0019 No MD 34 NA 32 treatment 045-013-0021 No ND ND NA NDtreatment 045-014-1001 No MD MD NA MD treatment ^(a)Baseline is theclosest screening measurement before the randomization date. ^(b)Fortreated patients, the assessment was performed after the device had beenimplanted, but prior to first idursulfase-IT dose. Abbreviations: BI =broad independence; MD = missing data, either test was not attempted orchild could not cooperate; NA = not applicable; ND = number notderivable

To understand whether there was a relationship between the cognitivechanges and the behavioral aspects of daily living, the correlationcoefficients were calculated between the DAS-II General ConceptualAbility and the overall Broad Independence Score of the SIB-R, as wellas the subdomains (see Table 12).

TABLE 12 Summary of Correlations between Selected Cognitive Tests - ITTPopulation Correlation Parameter 1 Parameter 2 Coefficient DAS-II GCAStandard DAS-II SNC Standard Scores 0.9482 Scores DAS-II GCA StandardSIB-R Broad Independence 0.8087 Scores Standard Scores DAS-II GCAStandard SIB-R Personal Living Skills 0.5081 Scores Standard ScoresDAS-II GCA Standard SIB-R Community Living Skills 0.7473 Scores StandardScores DAS-II GCA Standard SIB-R Social Interaction/ 0.7060 ScoresCommunication Skills Standard Scores DAS-II GCA Standard SIB-R MotorSkills Standard 0.5467 Scores Scores Note: Mixed models were utilized toaccount for repeated measurements.

As can be seen in Table 12, the DAS-II GCA and SIB-R BI standard scoreswere well correlated (r=0.8087), and good correlations were also seenbetween the GCA and standard scores for other SIB-R subdomains which,collectively, gauge a child's ability to function independently. Thesecorrelations also suggest that the cognitive improvements observed aremore than academic in value and truly translate as a measure ofimprovement in the child's ability to function independently. These highcorrelation numbers constitute an important aspect of the validation ofthe DAS-II as clinically relevant measure in the MPS II population.

Taken together, these data demonstrate that intrathecal administrationof recombinant I2S enzyme can effectively treat cognitive impairment inHunter syndrome patients. It is expected that longer duration oftreatment and/or early intervention in the trajectory ofneurodevelopmental decline with idursulfase-IT may be particularlyuseful in improving cognitive performance by, e.g., stabilizing orincreasing the DAS-II score in children with cognitive impairment due toHunter Syndrome.

While certain compounds, compositions and methods described herein havebeen described with specificity in accordance with certain embodiments,the following examples serve only to illustrate the compounds of theinvention and are not intended to limit the same.

The articles “a” and “an” as used herein in the specification and in theclaims, unless clearly indicated to the contrary, should be understoodto include the plural referents. Claims or descriptions that include“or” between one or more members of a group are considered satisfied ifone, more than one, or all of the group members are present in, employedin, or otherwise relevant to a given product or process unless indicatedto the contrary or otherwise evident from the context. The inventionincludes embodiments in which exactly one member of the group is presentin, employed in, or otherwise relevant to a given product or process.The invention also includes embodiments in which more than one, or theentire group members are present in, employed in, or otherwise relevantto a given product or process. Furthermore, it is to be understood thatthe invention encompasses all variations, combinations, and permutationsin which one or more limitations, elements, clauses, descriptive terms,etc., from one or more of the listed claims is introduced into anotherclaim dependent on the same base claim (or, as relevant, any otherclaim) unless otherwise indicated or unless it would be evident to oneof ordinary skill in the art that a contradiction or inconsistency wouldarise. Where elements are presented as lists, (e.g., in Markush group orsimilar format) it is to be understood that each subgroup of theelements is also disclosed, and any element(s) can be removed from thegroup. It should be understood that, in general, where the invention, oraspects of the invention, is/are referred to as comprising particularelements, features, etc., certain embodiments of the invention oraspects of the invention consist, or consist essentially of suchelements, features, etc. For purposes of simplicity those embodimentshave not in every case been specifically set forth in so many wordsherein. It should also be understood that any embodiment or aspect ofthe invention can be explicitly excluded from the claims, regardless ofwhether the specific exclusion is recited in the specification. Thepublications, websites and other reference materials referenced hereinto describe the background of the invention and to provide additionaldetail regarding its practice are hereby incorporated by reference,

We claim:
 1. A method of treating Hunter Syndrome comprising a step ofadministering intrathecally to a subject in need of treatment arecombinant iduronate-2-sulfatase (I2S) enzyme at a therapeuticallyeffective dose and an administration interval for a treatment periodsufficient to improve, stabilize or reduce declining of one or morecognitive, adaptive, motor, and/or executive functions relative to acontrol. 2-7. (canceled)
 8. The method of claim 1, wherein thetherapeutically effective dose, once administered regularly at theadministration interval, results in maximum serum concentration(C_(max)) of the recombinant I2S enzyme within a range fromapproximately 50 to about 300 ng/mL. 9-15. (canceled)
 16. The method ofclaim 1, wherein the intrathecal administration is through intermittentor continuous access to an implanted intrathecal drug delivery device(IDDD).
 17. The method of claim 16, wherein the intrathecaladministration is through continuous access to the implanted IDDD forgreater than about 0.5, 1.0, 1.5, or 2 hours. 18-24. (canceled)
 25. Themethod of claim 1, wherein the one or more cognitive, adaptive, motor,and/or executive functions are assessed by Bayley Scales of InfantDevelopment Version III (BSID-III).
 26. The method of claim 24, whereinthe intrathecal administration of the recombinant I2S enzyme results inimproved general conceptual ability (GCA) score or BSID-IIIdevelopmental quotient relative to the control.
 27. (canceled)
 28. Themethod of claim 25, wherein the intrathecal administration of therecombinant I2S enzyme results in stabilization of the GCA score orBSID-III developmental quotient relative to the control.
 29. The methodof claim 28, wherein the intrathecal administration of the recombinantI2S enzyme results in stabilization of the GCA score or BSID-IIIdevelopmental quotient for more than about 6 months. 30-32. (canceled)33. The method of claim 1, wherein the intrathecal administration of therecombinant I2S enzyme further results in improvement or stabilizationof one or more adaptive functions assessed by the Scales of IndependentBehavior-Revised (SIB-R).
 34. The method of claim 1, wherein theintrathecal administration of the recombinant I2S enzyme further resultsin improvement or stabilization of one or more executive functionsassessed by the Behavior Rating Inventory of Executive Function®(BRIEF®). 35-40. (canceled)
 41. The method of claim 1, wherein theintrathecal administration is performed in conjunction with intravenousadministration of the recombinant I2S enzyme. 42-45. (canceled)
 46. Themethod of claim 41, wherein the intravenous administration of therecombinant I2S enzyme is at a dose of about 0.5 mg/kg body weight.47-50. (canceled)
 51. A method of treating Hunter syndrome comprisingadministering intrathecally to a subject in need of treatment arecombinant iduronate-2-sulfatase (I2S) enzyme at a firsttherapeutically effective dose; and administering intravenously to thesubject the recombinant I2S enzyme at a second therapeutically effectivedose for a treatment period sufficient to improve, stabilize or reducedeclining of one or more cognitive, adaptive, motor, and/or executivefunctions relative to a control.
 52. A method of treating HuntersSyndrome comprising a step of administering intrathecally to a subjectin need of treatment a recombinant iduronate-2-sulfatase (I2S) enzyme ata therapeutically effective dose and an administration interval for atreatment period sufficient to decrease glycosaminoglycan (GAG) level inthe cerebrospinal fluid (CSF) relative to a control. 53-60. (canceled)61. The method of claim 52, wherein the administration interval ismonthly.
 62. (canceled)
 63. The method of claim 52, wherein theadministration interval is once every two, three, four, five, six ormore months 64-71. (canceled)
 72. The method of claim 51, wherein thetreatment period is at least 6 months.
 73. (canceled)
 74. The method ofclaim 52, wherein the intrathecal administration of the recombinant I2Senzyme results in the GAG level in the CSF lower than 1000 ng/ml. 75-77.(canceled)
 78. The method of claim 52, wherein the subject in need oftreatment is at least 2 years old.
 79. The method of claim 52, whereinthe subject in need of treatment is younger than 2 years old. 80-106.(canceled)